Compositions and methods comprising aspartyl-trna synthetases having non-canonical biological activities

ABSTRACT

Isolated aspartyl-tRNA synthetase polypeptides and polynucleotides having non-canonical biological activities are provided, as well as compositions and methods related thereto.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/751,358, filed Mar. 31, 2010; which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/165,194,filed Mar. 31, 2009, which is incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ATYR-01202US_ST25_SEQ_LISTING.txt. The text fileis 13 KB, was created on May 4, 2015, and is being submittedelectronically via EFS-Web.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to forms of aspartyl-tRNAsynthetase (AspRS) polypeptides, compositions comprising suchpolypeptides, and methods of using same.

2. Description of the Related Art

Aminoacyl-tRNA synthetases, which catalyze the aminoacylation of tRNAmolecules, are essential for decoding genetic information during theprocess of translation. In higher eukaryotes, aminoacyl-tRNA synthetasesassociate with other polypeptides to form supramolecular multienzymecomplexes. Each of the eukaryotic tRNA synthetases consists of a coreenzyme, which is closely related to the prokaryotic counterpart of thetRNA synthetase, and an additional domain that is appended to theamino-terminal or carboxyl-terminal end of the core enzyme. Humantyrosyl-tRNA synthetase (TyrRS), for example, has a carboxyl-terminaldomain that is not part of prokaryotic and lower eukaryotic TyrRSmolecules.

Several aminoacyl-tRNA synthetases have been demonstrated to havenon-canonical functions distinct from their involvement in translation.For example, Mini-tyrosyl tRNA synthetase (mini-TyrRS), the N-terminaldomain of TyrRS which corresponds to amino acid residues 1-364 and iscleaved by polymorphonuclear cell elastase and plasmin, is a member ofthe aminoacyl tRNA synthetase “AARS” multifunction cytokine-likeproteins and peptides. In vitro, Mini-TyrRS has been shown to stimulateneutrophil activation and chemotaxis, endothelial cell proliferation andmigration, and is pro-angiogenic in chick chorioallantoic membrane (CAM)and mouse matrigel assays. Mini-TyrRS has an ELR motif that, likeCXC-chemokines such as IL-8, confers its chemokine and angiogenicactivities. Like other ELR-containing cytokines, mutation of this motifinhibits mini-TyrRS binding and stimulation of leukocytes andangiogenesis.

In addition, truncated forms or TrpRS have been demonstrated to haveangiogenic properties. In normal human cells, there are two forms ofTrpRS that can be detected: a major form consisting of the full-lengthmolecule (amino acid residues 1-471) and a minor truncated form. Theminor form is generated by the deletion of an amino-terminal domainthrough alternative splicing of the pre-mRNA. The amino-terminus ofminiTrpRS has been determined to be the methionine residue at position48 of the full-length TrpRS molecule. Alternatively, truncated TrpRS canbe generated by proteolysis. For example, bovine TrpRS is highlyexpressed in the pancreas and is secreted into the pancreatic juice,thus resulting in the production of a truncated TrpRS molecule.Additional studies indicate that mini-TrpRS inhibits VEGF-induced cellproliferation and migration (Wakasugi et al., Proc. Natl. Acad. Sci. 99:173-177 (2002)). In particular, a chick CAM assay shows that mini TrpRSblocks angiogenic activity of VEGF. In contrast, the full-length TrpRSdoes not inhibit angiogenesis. Thus, removal of the first 48 amino acidresidues exposes the anti-angiogenic activity of TrpRS. Therefore, aswith TyrRS, certain forms of TrpRS possess activities other than theaminoacylation of tRNA.

Given these observations of non-canonical and therapeutically relevantactivities associated alternative forms of TyrRS and TrpRS, there is aneed to identify biologically relevant forms and/or activities of otheraminoacyl-tRNA synthetase proteins in order to exploit the fulltherapeutic potential of this family of proteins. Accordingly, thepresent invention addresses these needs and offers other relatedadvantages.

SUMMARY OF THE INVENTION

The present invention stems from the discovery that certainaspartyl-tRNA synthetase (AspRS) polypeptides possess non-canonicalbiological activities of therapeutic relevance. Therefore, according toone aspect, the present invention provides isolated AspRS polypeptideshaving at least one non-canonical biological activity, as well activefragments and variants thereof which substantially retain saidnon-canonical activity. “Non-canonical” activity,” as used herein,refers generally to an activity possessed by a AspRS polypeptide of theinvention that is other than aminoacylation and, more specifically,other than the addition of aspartic acid onto a tRNA^(Asp) molecule. Asdetailed herein, in certain embodiments, a non-canonical biologicalactivity exhibited by a AspRS polypeptide of the invention may include,but is not limited to, modulation of cell proliferation, modulation ofapoptosis, modulation of inflammation, modulation of celldifferentiation, modulation of angiogenesis, modulation of cell binding,modulation of Akt-mediated cell signaling, modulation of cellularmetabolism, modulation of cytokine production or activity, andmodulation of toll-like receptor signaling, and the like.

In certain embodiments, the AspRS polypeptide of the invention is acontiguous fragment of a full length mammalian AspRS protein. In a morespecific embodiment, the AspRS polypeptide is a contiguous fragment ofthe human AspRS protein sequence set forth in SEQ ID NO: 1.Illustratively, the fragments may be of essentially any length, providedthey are not full length and further provided they retain at least onenon-canonical biological activity of interest. In certain illustrativeembodiments, a AspRS polypeptide of the invention will range in sizefrom about 20-50, 20-100, 20-200, 20-300, 20-400, or 20-500 amino acidsin length. In other embodiments, the AspRS polypeptide of the inventionwill range in size from about 50-100, 50-200, 50-300, 50-400, or 50-500amino acids in length. In other embodiments, the AspRS polypeptide ofthe invention will range in size from about 100-200, 100-300, 100-400,or 100-500 amino acids in length. In still other illustrativeembodiments, the AspRS polypeptide of the invention will range in sizefrom about 200-300, 200-400, or 200-500 amino acids in length.

In further embodiments of the invention, an AspRS polypeptide comprisesan active variant (i.e., retains at least one non-canonical biologicalactivity of interest) of a fragment of an AspRS protein sequence, suchas the human AspRS protein sequence set forth in SEQ ID NO: 1. In a morespecific embodiment, the active variant is a polypeptide having at least70%, 80%, 90%, 95% or 99% identity along its length to a humanaspartyl-tRNA synthetase sequence set forth in SEQ ID NO: 1.

Other embodiments of the invention provide AspRS splice variants andpoint mutants, whether naturally or non-naturally occurring, thatpossess one or more non-canonical activities. In certain embodiments,the AspRS comprises an amphiphilic helix domain.

In a more specific embodiment of the invention, an AspRS polypeptidecomprises a fragment of the human AspRS sequence of SEQ ID NO: 1,consisting essentially of amino acid residues 1-154, 1-171, 1-174, 1-31,399-425, 413-476 or 397-425, or an active fragment or variant thereofthat substantially retains at least one non-canonical biologicalactivity of interest.

In other specific embodiments, the AspRS polypeptide is not apolypeptide as set forth in NCBI Accession No. NP001340.

According to another aspect of the invention, there are provided fusionproteins comprising at least one AspRS polypeptide as described hereinand a heterologous fusion partner.

According to another aspect of the invention, there are providedisolated polynucleotides encoding the polypeptides and fusion proteinsas described herein, as well as expression vectors comprising suchpolynucleotides, and host cell comprising such expression vectors. Alsoincluded are oligonucleotides that specifically hybridize to an AspRSpolynucleotide. In certain embodiments, the oligonucleotide is a primer,a probe, or an antisense oligonucleotide. Other embodiments relate toRNAi agents that target an AspRS polynucleotide.

According to another aspect of the invention, there are provided bindingagents (e.g., antibodies and antigen-binding fragments thereof) thathave binding specificity for an AspRS polypeptide of the invention, orone of its cellular binding partners. In certain embodiments, thebinding agent is an antibody, an antigen-binding fragment thereof, apeptide, a peptide mimetic, a small molecule, or an aptamer. In someembodiments, the binding agent antagonizes a non-canonical activity ofthe AspRS polypeptide. In other embodiments, the binding agent agonizesa non-canonical activity of the AspRS polypeptide.

According to yet another aspect of the invention, there are providedcompositions, e.g., pharmaceutical compositions, comprisingphysiologically acceptable carriers and at least one of the isolatedpolypeptides, fusion proteins, binding agents such as antibodies,isolated polynucleotides, expression vectors, host cells, etc., of theinvention, as described herein.

Certain embodiments relate to methods of determining presence or levelsof an AspRS polypeptide in a sample, comprising contacting the samplewith one or binding agents that specifically bind to an AspRSpolypeptide as described herein, detecting the presence or absence ofthe binding agent, and thereby determining the presence or levels of theAspRS polypeptide. Certain embodiments include methods of determiningpresence or levels of an AspRS polypeptide in a sample, comprisingintroducing the sample into a molecular detector that is capable ofspecifically identifying an AspRS polypeptide as described herein, andthereby determining the presence or levels of the AspRS polypeptide. Inspecific embodiments, the molecular detector is a mass spectrometer(MS). Certain embodiments include comparing the presence or levels ofthe AspRS protein fragment to a control sample or a predetermined value.Some embodiments include characterizing the state of the sample todistinguish it from the control. In specific embodiments, the sample andcontrol comprise a cell or tissue, and the method comprisesdistinguishing between cells or tissues of different species, cells ofdifferent tissues or organs, cells at different cellular developmentalstates, cells at different cellular differentiation states, or healthyand diseased cells.

Also included are methods of identifying a compound that specificallybinds to an AspRS polypeptide, or one or more of its cellular bindingpartners, comprising a) combining the AspRS polypeptide or its cellularbinding partner or both with at least one test compound under suitableconditions, and b) detecting binding of the AspRS polypeptide or itscellular binding partner or both to the test compound, therebyidentifying a compound that specifically binds to the AspRS polypeptideor its cellular binding partner or both. In certain embodiments, thetest compound is a polypeptide or peptide, an antibody orantigen-binding fragment thereof, a peptide mimetic, or a smallmolecule. In some embodiments, the test compound agonizes anon-canonical biological activity of the AspRS polypeptide or itscellular binding partner. In other embodiments, the test compoundantagonizes a non-canonical biological activity of the AspRS polypeptideor its cellular binding partner. Also included are compounds identifiedby any of the methods provided herein.

Also provided by the present invention, in other aspects, are methodsfor modulating a cellular activity by contacting a cell or tissue with acomposition of the invention, as described herein, wherein the cellularactivity to be modulated is selected from the group consisting of cellmigration, cell proliferation, apoptosis, inflammation, celldifferentiation, angiogenesis, modulation of cell binding, Akt-mediatedcell signaling, cellular metabolism, cytokine production, and toll-likereceptor signaling, and the like. In certain embodiments, the cellularactivity is cytokine production. In specific embodiments, the cytokineis any one or more of IL1-β, IL-6, IL-8, IL-10, IL-12p40, MIP1-α,MIP-1β, GRO-α, MCP-1, or IL-1ra. In some embodiments, the cellularactivity is toll-like receptor (TLR) signaling. In particularembodiments, the TLR is TLR2, TLR4, or both. Certain embodiments includemethods of stimulating an innate immune response. In some embodiments,the cell is in a subject.

In other aspects, the present invention provides methods for treating adisease, disorder or other condition in a subject in need thereof byadministering a composition according to the present invention. By wayof illustration, such diseases, disorders or conditions may include, butare not limited to, inflammatory diseases, autoimmune diseases,neoplastic diseases (e.g., cancers), metabolic diseases, neurologicaldiseases, infections, cardiovascular diseases, and diseases associatedwith abnormal angiogenesis.

BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS

SEQ ID NO: 1 is the full length amino acid sequence of humanaspartyl-tRNA synthetase (AspRS).

SEQ ID NO: 2 is a nucleic acid sequence encoding the AspRS polypeptideof SEQ ID NO: 1.

SEQ ID NO:3 is the amino acid sequence of a 32 amino acid human AspRSpeptide.

SEQ ID NO:4 is the amino acid sequence of a 32 amino acid rat AspRSpeptide.

SEQ ID NO:5 is a consensus sequence of the positively charged residuesof the AspRS amphiphilic helix.

SEQ ID NO:6 is the amino acid sequence of a portion of an anophelesmosquito AspRS N-terminal helix.

SEQ ID NO:7 is the amino acid sequence of a portion of a deer tick AspRSN-terminal helix.

SEQ ID NO:8 is the amino acid sequence of a portion of an owl limpetAspRS N-terminal helix.

SEQ ID NO:9 is the amino acid sequence of a portion of a leach AspRSN-terminal helix.

SEQ ID NO:10 is the amino acid sequence of a portion of a Xenopus AspRSN-terminal helix.

SEQ ID NO:11 is the amino acid sequence of a portion of a Japanesepuffer fish AspRS N-terminal helix.

SEQ ID NO:12 is the amino acid sequence of a portion of a green spottedpuffer AspRS N-terminal helix.

SEQ ID NO:13 is the amino acid sequence of a portion of the sticklebackAspRS N-terminal helix.

SEQ ID NO:14 is the amino acid sequence of a portion of a chicken AspRSN-terminal helix.

SEQ ID NO:15 is the amino acid sequence of a portion of a bovine AspRSN-terminal helix.

SEQ ID NO:16 is the amino acid sequence of a portion of a rat AspRSN-terminal helix.

SEQ ID NO:17 is the amino acid sequence of a portion of a mouse AspRSN-terminal helix.

SEQ ID NO:18 is the amino acid sequence of a portion of a rock hyraxAspRS N-terminal helix.

SEQ ID NO:19 is the amino acid sequence of a portion of an opossum AspRSN-terminal helix.

SEQ ID NO:20 is the amino acid sequence of a portion of a tarsier AspRSN-terminal helix.

SEQ ID NO:21 is the amino acid sequence of a portion of an orangutanAspRS N-terminal helix.

SEQ ID NO:22 is the amino acid sequence of a portion of a chimpanzeeAspRS N-terminal helix.

SEQ ID NO:23 is the amino acid sequence of a portion of a human AspRSN-terminal helix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show (A) the domain structure and (B) amino acid sequence ofAspRS (SEQ ID NO:1), and (C and D) illustrate the SDS-PAGE separation offragments of AspRS generated by controlled proteolysis of thefull-length AspRS protein with human neutrophil elastase. FIG. 1C is anSDS-PAGE gel, 4-12% MOPS, showing full-length AspRS and digestion withPMN elastase. FIG. 1D is an SDS-PAGE gel, 12% MES, showing full-lengthAspRS and digestion with PMN elastase.

FIGS. 2A-2B demonstrate the activation of Akt in endothelial cells(bAEC) treated with AspRS (also referred to as DRS) fragments of theinvention. FIG. 2A shows phosphorylation of Akt induced by treatmentwith pool of elastase generated AspRS fragments, and FIG. 2B shows atime course of Akt phosphorylation by cut pools of AspRS.

FIG. 3 shows the increased secretion of TNF-α by peripheral bloodmononuclear cells (PBMCs) treated with an AspRS fragment of theinvention, D1, in comparison to TNF-α secretion by PBMCs treated witheither full-length AspRS (DRS) or the positive control,endothelial-monocyte-activating polypeptide II (EMAP). PBMCs weretreated for 24 hours with D1, DRS, or EMAP II protein and assayed forTNF-α secretion.

FIG. 4 shows illustrative cytokines that are secreted followingtreatment of PBMCs with AspRS fragment D1. PBMCs were treated for 24hours, assayed for secretion of 27 different cytokines, and found toincrease secretion of IL1-β, IL-6, IL-8, IL-10, IL-12p40, MIP1-α,MIP-1β, GRO-α, MCP-1, and IL-1ra.

FIG. 5 shows that the AspRS fragment D1 activates monocytes in a celltype specific manner. PBMCs were treated for 24 hours with PBS as anegative control, PHA as a positive control, and AspRS fragment D1, andassayed to detect cell-surface markers of activation on monocytes andlymphocytes.

FIG. 6 shows that the AspRS fragment D1 induces secretion of TNF-α frommonocyte (e.g., THP-1) and macrophage (e.g., RAW 267.7) cell lines.

FIG. 7 shows that the AspRS fragment D1 induces chemotaxis of amacrophage cell line. FIG. 7A shows the experimental set-up to assaycell migration using a boudin chamber, and FIG. 1B shows that RAW 264.7macrophage cells migrate in a dose-dependent manner towards the D1fragment.

FIG. 8 shows that the TNF-α secretion mediated by the AspRS fragment D1in THP-1 monocytes is inhibited by an inhibitor of MEK (U0126), a keycomponent in the MAP kinase signaling pathway, but not by an inhibitorof PI3 kinase signaling (LY294022). LPS is used as a positive controland its activity is blocked by both inhibitors.

FIG. 9 shows that the AspRS fragment D1 inhibits VEGF-inducedangiogenesis. Matrigel solutions containing PBS, sutent, or D1 fragmentin combination with VEGF were implanted into mice and analyzed for newblood vessel infiltration into the matrigel plug.

FIG. 10 shows the results of an experiment which suggests that theN-terminal region of the AspRS fragment D1 is responsible for itscytokine activity. The presence of a 6×his affinity tag on theN-terminus of D1, as compared to on the C-terminus of D1, reduces theTNF-α secretion activity of the fragment.

FIG. 11 shows that the AspRS fragment D1 contains a mammalian-specific32 amino acid sequence at its N-terminus SEQ ID NO:3 is the human AspRS32 amino acid peptide and SEQ ID NO:4 is the rat AspRS 32 amino acidpeptide. A 32 amino acid peptide found only at the N-terminus ofmammalian AspRS, and not found in yeast AspRS, is dispensable forcanonical tRNA synthetase activity and predicted to contain a putativehelix (see Jacobo-Molina and Yang (1989); and Escalante and Yang, JBC(1992)).

FIGS. 12A-12C show the identification, evolution and crystallization ofhuman AspRS fragment D1. FIG. 12A shows the steps by which RAW264.7mouse macrophages were subjected to SDS-PAGE analysis; protein bandswere cut out and analyzed by LC MS/MS and an N-terminal fragment ofAspRS was identified as D1. FIG. 12B shows that the appended N-terminusof AspRS is an evolved domain. FIG. 12C shows the crystal structure offull length dimeric human AspRS solved to a resolution of 1.9 Å; theN-terminal tRNA anticodon-binding domain, the aminoacylation domain, andthe 30 amino acid linker connecting the D1 fragment and theaminoacylation domain are indicated.

FIGS. 13A-13C show that D1 induces pro-inflammatory andanti-inflammatory cytokine secretion in vivo and in vitro. FIG. 13Ashows in vivo TNF-α and IL-10 serum levels from mice injectedintravenously with 10 mg/kg D1. Mice show an increase in TNF-α after 2hours that is quickly cleared by 6 hours while IL-10 levels continue toincrease. FIG. 13B shows in vitro TNF-α & IL-10 release from PBMCs after4 & 24 hours respectively. Cells show an increase with D1 (250 nM)treatment but not with full-length AspRS (250 nM); LPS (10 EU) alsoshows a strong TNF-α response. The flow cytometry analysis in FIG. 13Creveals D1 binding to 83% of primary monocytes and 14% of the totallymphocyte population. Within primary lymphocytes, D1 binds to 76% ofCD19+ B-cells.

FIGS. 14A-14C show that D1 activates NF-kB via toll-like receptors 2 and4 (TLR2 and TLR4). FIG. 14A shows that D1 activates NF-kB in RAW264.7mouse macrophages; RAW-Blue cells encoding an NF-κB-inducible secretedembryonic alkaline phosphatase reporter gene showed a dose dependentactivation of NF-κB with D1 as compared to the lack of activation byAspRS. As shown in FIG. 14B, D1 (1 μM) activates both TLR2 and TLR4over-expressing HEK293 cells whereas AspRS (1 μM) did not show activity;stably transfected HEK293 cells expressing either TLR2 or TLR4 with anNF-κB-inducible reporter demonstrated that D1 can induce NF-κBactivation. In FIG. 14C, flow cytometry shows that D1 binds HEK cellsover-expressing TLR2 or TLR4, but does not bind to control cells.

FIGS. 15A-15D show a characterization of D1 activity, relating in partto the N-terminal amphiphilic helix. The helical wheel in FIG. 15Adepicts the N-terminus of human AspRS, and reveals an amphiphilic helix.The alignment in FIG. 15B (SEQ ID NOS:6-23, from top to bottom)illustrates two D1 mutants that were designed in view of the N-terminalhelix; a triple alanine (AAA) mutation to neutralize the negativelycharged residues, and a partial charge reversal mutant (SKK) thatrepresents the yeast sequence. FIG. 15C shows an increase in in vitroTNF-α & IL-10 release from PBMCs after 24 treatment with D1 (50 nM) butnot with full-length AspRS (50 nM) or with the Δ22 mutant (50 nM); thecharge mutants (AAA and SKK) also show decreased activity. FIG. 15Dillustrates how D1 can be released from macrophage cells and binds tomonocytes, T-cells and B-cells via TLR2 and TLR4 receptors to elicit anearly proinflammatory response of TNF-α release, followed by ananti-inflammatory response of IL-10 release.

FIGS. 16A-16C show that D1 activity is not due to endotoxincontamination. In FIG. 16A, mammalian expressed D1 induces cytokinesecretion in peripheral blood mononuclear cells. D1 was expressed with aconventional secretion sequence in HEK293 cells. Conditioned mediacontaining secreted D1 was collected, concentrated and incubated withPBMC; D1 containing media induced TNF-α release which was not observedin mock transfected media. As shown in FIG. 16B, D1 activity isindependent of endotoxin contamination; D1 cytokine release wasunaltered in the presence of polymyxin B, an inactivator of endotoxin,whereas lipopolysaccharide (LPS) was completely inhibited. FIG. 16Cshows that D1 digestion by proteinase K abolishes PBMC cytokinestimulating ability. D1 was digested completely by overnight treatmentwith proteinase K, and digested D1 was added to PBMC and TNF-α secretionwas measured by ELISA.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of molecular biologyand recombinant DNA techniques within the skill of the art, many ofwhich are described below for the purpose of illustration. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); A Practical Guide to Molecular Cloning (B. Perbal,ed., 1984).

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. By way of example, “an element” means oneelement or more than one element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

An “agonist” refers to a molecule that intensifies or mimics thenon-canonical biological activity of an AspRS. Agonists may includeproteins, nucleic acids, carbohydrates, small molecules, or any othercompound or composition that modulates the activity of an AspRS eitherby directly interacting with the AspRS or its binding partner, or byacting on components of the biological pathway in which the AspRSparticipates. Included are partial and full agonists.

The term “antagonist” refers to a molecule that inhibits or attenuatesthe non-canonical biological activity of an AspRS. Antagonists mayinclude proteins such as antibodies, nucleic acids, carbohydrates, smallmolecules, or any other compound or composition that modulates theactivity of an AspRS or its binding partner, either by directlyinteracting with the AspRS or its binding partner or by acting oncomponents of the biological pathway in which the AspRS participates.Included are partial and full antagonists.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

By “gene” is meant a unit of inheritance that occupies a specific locuson a chromosome and consists of transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (i.e., introns, 5′ and 3′ untranslated sequences).

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395), which is incorporatedherein by reference. In this way sequences of a similar or substantiallydifferent length to those cited herein could be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

The term “host cell” includes an individual cell or cell culture thatcan be or has been a recipient of any recombinant vector(s) or isolatedpolynucleotide of the invention. Host cells include progeny of a singlehost cell, and the progeny may not necessarily be completely identical(in morphology or in total DNA complement) to the original parent celldue to natural, accidental, or deliberate mutation and/or change. A hostcell includes cells transfected or infected in vivo or in vitro with arecombinant vector or a polynucleotide of the invention. A host cellwhich comprises a recombinant vector of the invention is a recombinanthost cell.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide,” as used herein, includes apolynucleotide that has been purified from the sequences that flank itin its naturally-occurring state, e.g., a DNA fragment which has beenremoved from the sequences that are normally adjacent to the fragment.Alternatively, an “isolated peptide” or an “isolated polypeptide” andthe like, as used herein, includes the in vitro isolation and/orpurification of a peptide or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell; i.e., it is not significantly associated with in vivo substances.

The term “mRNA” or sometimes refer by “mRNA transcripts” as used herein,include, but not limited to pre-mRNA transcript(s), transcriptprocessing intermediates, mature mRNA(s) ready for translation andtranscripts of the gene or genes, or nucleic acids derived from the mRNAtranscript(s). Transcript processing may include splicing, editing anddegradation. As used herein, a nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript or a subsequence thereof has ultimately served as a template.A cDNA reverse transcribed from an mRNA, an RNA transcribed from thatcDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, etc., are all derived from the mRNA transcript anddetection of such derived products is indicative of the presence and/orabundance of the original transcript in a sample. Thus, mRNA derivedsamples include, but are not limited to, mRNA transcripts of the gene orgenes, cDNA reverse transcribed from the mRNA, cRNA transcribed from thecDNA, DNA amplified from the genes, RNA transcribed from amplified DNA,and the like.

“Non-canonical” activity as used herein, refers generally to an activitypossessed by an AspRS polypeptide of the invention that is other thanaminoacylation and, more specifically, other than the addition of itscognate amino acid onto its cognate tRNA molecule. Non-limiting examplesof non-canonical activities include RNA-binding, amino acid-binding,modulation of cell proliferation, modulation of cell migration,modulation of cell differentiation (e.g., hematopoiesis), modulation ofapoptosis or other forms of cell death, modulation of cell signaling,modulation of angiogenesis, modulation of cell binding, modulation ofcellular metabolism, modulation of cytokine production or activity,modulation of cytokine receptor activity, modulation of inflammation,and the like.

The term “modulating” includes “increasing” or “stimulating,” as well as“decreasing” or “reducing,” typically in a statistically significant ora physiologically significant amount as compared to a control. An“increased” or “enhanced” amount is typically a “statisticallysignificant” amount, and may include an increase that is 1.1, 1.2, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times)(including all integers and decimal points in between and above 1, e.g.,1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no composition (theabsence of an agent or compound) or a control composition. A “decreased”or reduced amount is typically a “statistically significant” amount, andmay include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amountproduced by no composition (the absence of an agent or compound) or acontrol composition, including all integers in between. Other examplesof “statistically significant” amounts are described herein.

By “obtained from” is meant that a sample such as, for example, apolynucleotide extract or polypeptide extract is isolated from, orderived from, a particular source of the subject. For example, theextract can be obtained from a tissue or a biological fluid isolateddirectly from the subject. “Derived” or “obtained from” can also referto the source of a polypeptide or polynucleotide sequence. For instance,an AspRS sequence of the present invention may be “derived” from thesequence information of an AspRS proteolytic fragment or AspRS splicevariant, or a portion thereof, whether naturally-occurring orartificially generated, and may thus comprise, consist essentially of,or consist of that sequence.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

A “splice junction” as used herein includes the region in a mature mRNAtranscript or the encoded polypeptide where the 3′ end of a first exonjoins with the 5′ end of a second exon. The size of the region may vary,and may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 or more (including all integers in between) nucleotide or amino acidresidues on either side of the exact residues where the 3′ end of oneexon joins with the 5′ end of another exon. An “exon” refers to anucleic acid sequence that is represented in the mature form of an RNAmolecule after either portions of a precursor RNA (introns) have beenremoved by cis-splicing or two or more precursor RNA molecules have beenligated by trans-splicing. The mature RNA molecule can be a messengerRNA or a functional form of a non-coding RNA such as rRNA or tRNA.Depending on the context, an exon can refer to the sequence in the DNAor its RNA transcript. An “intron” refers to a non-coding nucleic acidregion within a gene, which is not translated into a protein. Non-codingintronic sections are transcribed to precursor mRNA (pre-mRNA) and someother RNAs (such as long noncoding RNAs), and subsequently removed bysplicing during the processing to mature RNA.

A “splice variant” refers to a mature mRNA and its encoded protein thatare produced by alternative splicing, a process by which the exons ofthe RNA (a primary gene transcript or pre-mRNA) are reconnected inmultiple ways during RNA splicing. The resulting different mRNAs may betranslated into different protein isoforms, allowing a single gene tocode for multiple proteins.

A “subject,” as used herein, includes any animal that exhibits asymptom, or is at risk for exhibiting a symptom, which can be treated ordiagnosed with an AspRS polynucleotide or polypeptide of the invention.Suitable subjects (patients) include laboratory animals (such as mouse,rat, rabbit, or guinea pig), farm animals, and domestic animals or pets(such as a cat or dog). Non-human primates and, preferably, humanpatients, are included.

“Treatment” or “treating,” as used herein, includes any desirable effecton the symptoms or pathology of a disease or condition that can beeffected by the non-canonical activities of an AspRS polynucleotide orpolypeptide, as described herein, and may include even minimal changesor improvements in one or more measurable markers of the disease orcondition being treated. Also included are treatments that relate tonon-AspRS therapies, in which an AspRS sequence described hereinprovides a clinical marker of treatment. “Treatment” or “treating” doesnot necessarily indicate complete eradication or cure of the disease orcondition, or associated symptoms thereof. The subject receiving thistreatment is any subject in need thereof. Exemplary markers of clinicalimprovement will be apparent to persons skilled in the art.

By “vector” or “nucleic acid construct” is meant a polynucleotidemolecule, preferably a DNA molecule derived, for example, from aplasmid, bacteriophage, yeast or virus, into which a polynucleotide canbe inserted or cloned. A vector preferably contains one or more uniquerestriction sites and can be capable of autonomous replication in adefined host cell including a target cell or tissue or a progenitor cellor tissue thereof, or be integrable with the genome of the defined hostsuch that the cloned sequence is reproducible. Accordingly, the vectorcan be an autonomously replicating vector, i.e., a vector that exists asan extra-chromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a linear or closed circular plasmid, anextra-chromosomal element, a mini-chromosome, or an artificialchromosome. The vector can contain any means for assuringself-replication. Alternatively, the vector can be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated.

The terms “wild-type” and “naturally occurring” are used interchangeablyto refer to a gene or gene product that has the characteristics of thatgene or gene product when isolated from a naturally occurring source. Awild-type gene or gene product (e.g., a polypeptide) is that which ismost frequently observed in a population and is thus arbitrarilydesigned the “normal” or “wild-type” form of the gene.

Aspartyl-tRNA Synthetase Polypeptides

The present invention relates generally to isolated AspRS polypeptides,polynucleotides encoding such polypeptides, binding agents that bindsuch polypeptides, analogs, variants and fragments of such polypeptides,etc., as well as compositions and methods of using any of the foregoing.Therefore, according to one aspect of the invention, there are providedAspRS polypeptides having non-canonical activities of therapeuticrelevance, as well as compositions comprising the same.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues andto variants and synthetic analogues of the same. Thus, these terms applyto amino acid polymers in which one or more amino acid residues aresynthetic non-naturally occurring amino acids, such as a chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally-occurring amino acid polymers.

Polypeptides are not limited to a specific length, but, in the contextof the present invention, typically represent a fragment of a fulllength protein, and may include post-translational modifications, forexample, glycosylations, acetylations, phosphorylations and the like, aswell as other modifications known in the art, both naturally occurringand non-naturally occurring. Polypeptides and proteins of the inventionmay be prepared using any of a variety of well known recombinant and/orsynthetic techniques, illustrative examples of which are furtherdiscussed below.

The recitation “polypeptide variant” refers to polypeptides that aredistinguished from a reference AspRS polypeptide (e.g., SEQ ID NO: 1, orany of its fragments such as D1, including fragments that consist ofamino acid residues 1-154, 1-171, 1-174, 1-177, 1-31, 399-425, 413-476or 397-425 of SEQ ID NO:1) by the addition, deletion, and/orsubstitution of at least one amino acid residue, and which typicallyretain at least one non-canonical activity, as described herein. Incertain embodiments, a polypeptide variant is distinguished from areference polypeptide by one or more substitutions, which may beconservative or nonconservative, as described herein and known in theart. In certain embodiments, the polypeptide variant comprisesconservative substitutions and, in this regard, it is well understood inthe art that some amino acids may be changed to others with broadlysimilar properties without changing the nature of the activity of thepolypeptide.

Polypeptide variants encompassed by the present invention will typicallyexhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity (determined as described below), alongtheir lengths, to the corresponding region of a wild-type mammalianAspRS protein, such as SEQ ID NO: 1, or any of its fragments such as D1,including fragments that consist of amino acid residues 1-154, 1-171,1-174, 1-177, 1-31, 399-425, 413-476 or 397-425 of SEQ ID NO:1. Alsoincluded are sequences differing from the reference AspRS sequences bythe addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150 or more amino acids but which retain theproperties of a reference AspRS polypeptide, such as a non-canonicalactivity. In certain embodiments, the amino acid additions or deletionsoccur at the C-terminal end and/or the N-terminal end of SEQ ID NO:1 orfragments thereof that consist of amino acid residues 1-154, 1-171,1-174, 1-177, 1-31, 399-425, 413-476 or 397-425 of SEQ ID NO:1. Incertain embodiments, the amino acid additions include 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or morewild-type residues (i.e., from the corresponding full-length AARSpolypeptide) that are proximal to the C-terminal end and/or theN-terminal end of these AspRS fragments.

In other embodiments, variant polypeptides differ from the correspondingAspRS reference sequences by at least 1% but less than 20%, 15%, 10% or5% of the residues. (If this comparison requires alignment, thesequences should be aligned for maximum similarity. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences.) The differences are, suitably, differences or changes at anon-essential residue or a conservative substitution.

Also included are biologically active “fragments” of the AspRS referencepolypeptides. Representative biologically active fragments generallyparticipate in an interaction, e.g., an intramolecular or aninter-molecular interaction. An inter-molecular interaction can be aspecific binding interaction or an enzymatic interaction. Aninter-molecular interaction can be between an AspRS polypeptide and acellular binding partner, such as a cellular receptor or other hostmolecule that participates in the non-canonical activity of the AspRSpolypeptide.

Typically, biologically active fragments comprise a domain or motif withat least one activity of an AspRS reference polypeptide and may includeone or more (and in some cases all) of the various active domains, andinclude fragments having a non-canonical activity. In some cases,biologically active fragments of an AspRS polypeptide have a biologicalactivity that is unique to the particular, truncated fragment, such thatthe full-length AspRS polypeptide may not have that activity. In certaincases, the biological activity may be revealed by separating thebiologically active AspRS polypeptide fragment from the otherfull-length AspRS polypeptide sequences, or by altering certain residuesof the full-length AspRS wild-type polypeptide sequence to unmask thebiologically active domains. For example, in certain illustrativeembodiments, an AspRS polypeptide may comprise all or a portion of anamphiphilic helix, as illustrated herein (see, e.g., SEQ ID NO:3),and/or a region of positive charged residues (see, e.g., SEQ ID NO:5).In certain embodiments, the amphiphilic helix is a 22 amino acid region,as described herein.

A biologically active fragment of an AspRS reference polypeptide can bea polypeptide fragment which is, for example, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 220, 240, 250, 260, 280, 300 or more contiguousor non-contiguous amino acids, including all integers in between, of theamino acid sequences set forth SEQ ID NO:1. In other illustrativeembodiments, an AspRS fragment of SEQ ID NO:1 may range in size fromabout 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-125,20-150 or 20-175 amino acids in length. In other embodiments, thefragment will range in size from about 30-40, 30-50, 30-60, 30-70,30-80, 30-90, 30-100, 30-125, 30-150 or 30-175 amino acids in length. Inother embodiments, the fragment will range in size from about 40-50,40-60, 40-70, 40-80, 40-90, 40-100, 40-125, 40-150 or 40-175 amino acidsin length. In still other illustrative embodiments, the fragment willrange in size from about 50-60, 50-70, 50-80, 50-90, 50-100, 50-125,50-150 or 50-175 amino acids in length.

In certain embodiments, the AspRS polypeptide is a truncated AspRSpolypeptide. A “truncated” AspRS, as used herein, refers to anaspartyl-tRNA synthetase protein which is shorter than its correspondingfull length AspRS protein, for example, due to removal of amino acidsfrom its N- and/or C-terminal ends. The extent of the truncation, thatis, the number of N- and/or C-terminal amino acid residues removed froma full length AspRS protein can vary considerably while still providingdesired cellular effects when administered to a cell, tissue or subject,as described herein. In certain embodiments, at least about 5, 10, 15,20, 25, 50, 75, 100, 150, 200, 250, 300, 350 amino acids, or more,including all intermediate lengths, are truncated from the N- and/orC-terminus of a full length mammalian AspRS protein. Intermediatelengths are intended to include all integers therebetween, for example,6, 7, 8, etc., 51, 52, 53, etc., 201, 202, 203, etc. Suitably, thebiologically-active fragment has no less than about 1%, 10%, 25%, or 50%of a non-canonical biologically-activity of an AspRS referencepolypeptide.

Also included are proteolytic fragments of an AspRS polypeptide, whichcan be characterized, identified, or derived according to a variety oftechniques. For instance, proteolytic fragments can be identified invitro, such as by incubating full-length or other AspRS polypeptideswith selected proteases, or they can be identified endogenously (i.e.,in vivo). In certain embodiments, protein fragments such as endogenousproteolytic fragments can be generated or identified, for instance, byrecombinantly expressing full-length or other AspRS polypeptides in aselected microorganism or eukaryotic cell that has been either modifiedto contain one or more selected proteases, or that naturally containsone or more proteases that are capable of acting on a selected AspRSpolypeptide, and isolating and characterizing the endogenously producedprotein fragments therefrom.

In certain embodiments, protein fragments such as endogenous (e.g.,naturally-occurring) proteolytic fragments can be generated oridentified, for instance, from various cellular fractions (e.g.,cytosolic, membrane, nuclear) and/or growth medium of variouscell-types, including, for example, macrophages such as RAW macrophages(e.g., RAW 264.7 macrophages), T-cells, including primary T-cells andT-cell lines such as Jurkats, and natural killer (NK) cells, amongothers. In certain embodiments, protein fragments such as endogenousproteolytic fragments, however generated, can be identified bytechniques such as mass-spectrometry, or equivalent techniques. Once anin vitro or endogenously identified protein fragment has been generatedor identified, it can be mapped or sequenced, and, for example, clonedinto an expression vector for recombinant production, or producedsynthetically.

A wide variety of proteases can be used to produce, identify, derive, orcharacterize the sequence of AspRS proteolytic fragments. Generally,proteases are usually classified according to three major criteria: (i)the reaction catalysed, (ii) the chemical nature of the catalytic site,and (iii) the evolutionary relationship, as revealed by the structure.General examples of proteases or proteinases, as classified by mechanismof catalysis, include aspartic proteases, serine proteases, cysteineproteases, and metalloproteases.

Most aspartic proteases belong to the pepsin family. This familyincludes digestive enzymes, such as pepsin and chymosin, as well aslysosomal cathepsins D and processing enzymes such as renin, and certainfungal proteases (e.g., penicillopepsin, rhizopuspepsin,endothiapepsin). A second family of aspartic proteases includes viralproteinases such as the protease from the AIDS virus (HIV), also calledretropepsin.

Serine proteases include two distinct families. First, the chymotrypsinfamily, which includes the mammalian enzymes such as chymotrypsin,trypsin, elastase, and kallikrein, and second, the substilisin family,which includes the bacterial enzymes such as subtilisin. The general 3Dstructure between these two families is different, but they have thesame active site geometry, and catalysis proceeds via the samemechanism. The serine proteases exhibit different substratespecificities, differences which relate mainly to amino acidsubstitutions in the various enzyme subsites (substrate residueinteracting sites). Some serine proteases have an extended interactionsite with the substrate whereas others have a specificity that isrestricted to the P1 substrate residue.

The cysteine protease family includes the plant proteases such aspapain, actinidin, and bromelain, several mammalian lysosomalcathepsins, the cytosolic calpains (calcium-activated), as well asseveral parasitic proteases (e.g., Trypanosoma, Schistosoma). Papain isthe archetype and the best studied member of the family. Recentelucidation of the X-ray structure of the Interleukin-1-beta ConvertingEnzyme has revealed a novel type of fold for cysteine proteinases.

The metalloproteases are one of the older classes of proteases, found inbacteria, fungi, and higher organisms. They differ widely in theirsequences and their 3D structures, but the great majority of enzymescontain a zinc atom that is catalytically active. In some cases, zincmay be replaced by another metal such as cobalt or nickel without lossof proteolytic activity. Bacterial thermolysin has been wellcharacterized and its crystallographic structure indicates that zinc isbound by two histidines and one glutamic acid. Many metalloproteasescontain the sequence motif HEXXH, which provides two histidine ligandsfor the zinc. The third ligand is either a glutamic acid (thermolysin,neprilysin, alanyl aminopeptidase) or a histidine (astacin, serralysin).

In certain illustrative embodiments, truncated AspRS polypeptides may beproduced using any of a variety of proteolytic enzymes using techniquesknown and available in the art. Illustrative proteases include, forexample, achromopeptidase, aminopeptidase, ancrod, angiotensinconverting enzyme, bromelain, calpain, calpain I, calpain II,carboxypeptidase A, carboxypeptidase B, carboxypeptidase G,carboxypeptidase P, carboxypeptidase W, carboxypeptidase Y, caspase 1,caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7,caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13,cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G,cathepsin H, cathepsin L, chymopapain, chymase, chymotrypsin,clostripain, collagenase, complement C1r, complement C1s, complementFactor D, complement factor I, cucumisin, dipeptidyl peptidase IV,elastase (leukocyte), elastase (pancreatic), endoproteinase Arg-C,endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C,enterokinase, factor Xa, ficin, furin, granzyme A, granzyme B, HIVProtease, IGase, kallikrein tissue, leucine aminopeptidase (general),leucine aminopeptidase (cytosol), leucine aminopeptidase (microsomal),matrix metalloprotease, methionine aminopeptidase, neutrase, papain,pepsin, plasmin, prolidase, pronase E, prostate specific antigen,protease alkalophilic from Streptomyces griseus, protease fromAspergillus, protease from Aspergillus saitoi, protease from Aspergillussojae, protease (B. licheniformis) (alkaline or alcalase), protease fromBacillus polymyxa, protease from Bacillus sp., protease from Rhizopussp., protease S, proteasomes, proteinase from Aspergillus oryzae,proteinase 3, proteinase A, proteinase K, protein C, pyroglutamateaminopeptidase, rennin, streptokinase, subtilisin, thermolysin,thrombin, tissue plasminogen activator, trypsin, tryptase and urokinase.

Certain embodiments relate to isolated AspRS polypeptides, comprising,consisting essentially of, or consisting of amino acid sequences thathave been derived from endogenous, naturally-occurring AspRS polypeptidefragments, and pharmaceutical compositions comprising said fragments,and methods of use thereof. In certain embodiments, as noted above, thesequences of AspRS protein fragments such as endogenous proteolyticfragments can be generated or identified, for instance, from variouscellular fractions (e.g., cytosolic, membrane, nuclear) and/orconditioned medium from various cell-types, including primary cells andcell lines. Examples of such cell types include, without limitation,immune cells such as monocytes, dendritic cells, macrophages (e.g., RAW264.7 macrophages; see Example 5), neutrophils, eosinophils, basophils,and lymphocytes, such as B-cells and T-cells (e.g., CD4+ helper and CD8+killer cells), including primary T-cells and T-cell lines such as JurkatT-cells, as well as natural killer (NK) cells.

In certain embodiments, AspRS protein fragments can be identified bytechniques such as mass-spectrometry, or equivalent techniques. Merelyby way of illustration and not limitation, in certain embodiments theproteomes from various cell types, tissues, or body fluids from avariety of physiological states (e.g., hyposia, diet, age, disease) orfractions thereof may be separated by 1D SDS-PAGE and the gel lanes cutinto bands at fixed intervals; after which the bands may be optionallydigested with an appropriate protease, such as trypsin, to release thepeptides, which may then be analyzed by 1D reverse phase LC-MS/MS. Theresulting proteomic data may be integrated into so-called peptographs,which plot, in the left panel, sequence coverage for a given protein inthe horizontal dimension (N to C terminus, left to right) versusSDS-PAGE migration in the vertical dimension (high to low molecularweight, top to bottom). The specific peptide fragments can then besequenced or mapped. In certain embodiments, the AspRS referencefragment may be characterized by its unique molecular weight, ascompared, for example, to the molecular weight of the correspondingfull-length AspRS.

As noted above, a polypeptide variant may differ from an AspRSpolypeptide of the invention in one or more substitutions, deletions,additions and/or insertions. Such variants may be naturally occurring ormay be synthetically generated, for example, by modifying one or more ofthe above polypeptide sequences of the invention and evaluating theirbiological activity as described herein using any of a number oftechniques well known in the art.

In other illustrative embodiments, the variant may be a splice variant,whether naturally or non-naturally occurring, wherein the polypeptidepossesses at least one non-canonical activity, e.g., as describedherein. In other illustrative embodiments, the variant contains one ormore point mutations relative to the wild type AspRS polypeptidesequence, whether naturally or non-naturally occurring, wherein thepolypeptide possesses at least one non-canonical activity, e.g., asdescribed herein.

In certain embodiments, a variant will contain conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. Modifications may be made in the structure ofthe polynucleotides and polypeptides of the present invention and stillobtain a functional molecule that encodes a variant or derivativepolypeptide with desirable characteristics. When it is desired to alterthe amino acid sequence of a polypeptide to create an equivalent, oreven an improved, variant of an AspRS polypeptide of the invention, oneskilled in the art, for example, can change one or more of the codons ofthe encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that generallydefines that protein's biological functional activity, certain aminoacid sequence substitutions can be made in a protein sequence, and, ofcourse, its underlying DNA coding sequence, and nevertheless obtain aprotein with like properties. It is thus contemplated that variouschanges may be made in the polypeptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode saidpolypeptides without appreciable loss of their desired utility oractivity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU  Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU  Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may also beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). For example, it is known that the relative hydropathiccharacter of the amino acid contributes to the secondary structure ofthe resultant protein, which in turn defines the interaction of theprotein with other molecules, for example, enzymes, substrates,receptors, DNA, antibodies, antigens, and the like. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, 1982). These values are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4);threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3);proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5);aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Asdetailed in U.S. Pat. No. 4,554,101, the following hydrophilicity valueshave been assigned to amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalentprotein. In such changes, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions may be based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl-methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onsecondary structure and hydropathic nature of the polypeptide.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polypeptide sequences, two sequences are said to be“identical” if the sequence of amino acids in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted, forexample, using the Megalign program in the Lasergene suite ofbioinformatics software (DNASTAR, Inc., Madison, Wis.), using defaultparameters. This program embodies several alignment schemes described inthe following references: Dayhoff, M. O. (1978) A model of evolutionarychange in proteins—Matrices for detecting distant relationships. InDayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp.345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp.626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego,Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers,E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb.Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425;Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—thePrinciples and Practice of Numerical Taxonomy, Freeman Press, SanFrancisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Nat'lAcad., Sci. USA 80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nucl. AcidsRes. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. BLAST and BLAST 2.0 can be used, for example with theparameters described herein, to determine percent sequence identity forthe polynucleotides and polypeptides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. For amino acid sequences, ascoring matrix can be used to calculate the cumulative score. Extensionof the word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one illustrative approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

In certain embodiments of the invention, there are provided fusionpolypeptides, and polynucleotides encoding fusion polypeptides. Fusionpolypeptides refer to AspRS polypeptides of the invention that have beencovalently linked, either directly or indirectly via an amino acidlinker, to one or more heterologous polypeptide sequences (fusionpartners). The polypeptides forming the fusion protein are typicallylinked C-terminus to N-terminus, although they can also be linkedC-terminus to C-terminus, N-terminus to N-terminus, or N-terminus toC-terminus. The polypeptides of the fusion protein can be in any order.

The fusion partner may be designed and included for essentially anydesired purpose provided they do not adversely affect the desiredactivity of the polypeptide. For example, in one embodiment, a fusionpartner comprises a sequence that assists in expressing the protein (anexpression enhancer) at higher yields than the native recombinantprotein. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

More generally, fusion to heterologous sequences, such as an Fcfragment, may be utilized to remove unwanted characteristics or toimprove the desired characteristics (e.g., pharmacokinetic properties)of an AspRS polypeptide. For example, fusion to a heterologous sequencemay increase chemical stability, decrease immunogenicity, improve invivo targeting, and/or increase half-life in circulation of an AspRSpolypeptide.

Fusion to heterologous sequences may also be used to createbi-functional fusion proteins, such as bi-functional proteins that arenot only possess a selected non-canonical activity through the AspRSpolypeptide, but are also capable of modifying (i.e., stimulating orinhibiting) other pathways through the heterologous polypeptide.Examples of such pathways include, but are not limited to, variousimmune system-related pathways, such as innate or adaptive immuneactivation pathways, or cell-growth regulatory pathways, such asangiogenesis. In certain aspects, the heterologous polypeptide may actsynergistically with the AspRS polypeptide to modulate a cellularpathway in a subject. Examples of heterologous polypeptides that may beutilized to create a bi-functional fusion protein include, but are notlimited to, thrombopoietin, cytokines (e.g., IL-11), chemokines, andvarious hematopoietic growth factors, in addition to biologically activefragments and/or variants thereof.

Fusion proteins may generally be prepared using standard techniques. Forexample, DNA sequences encoding the polypeptide components of a desiredfusion may be assembled separately, and ligated into an appropriateexpression vector. The 3′ end of the DNA sequence encoding onepolypeptide component is ligated, with or without a peptide linker, tothe 5′ end of a DNA sequence encoding the second polypeptide componentso that the reading frames of the sequences are in phase. This permitstranslation into a single fusion protein that retains the biologicalactivity of both component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures, ifdesired. Such a peptide linker sequence is incorporated into the fusionprotein using standard techniques well known in the art. Certain peptidelinker sequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No.4,751,180. The linker sequence may generally be from 1 to about 50 aminoacids in length. Linker sequences are not required when the first andsecond polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

In general, polypeptides and fusion polypeptides (as well as theirencoding polynucleotides) are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

In still other embodiments, an AspRS polypeptide of the invention may bepart of a dimer. Dimers may include, for example, homodimers between twoidentical AspRS polypeptides, heterodimers between two different AspRSpolypeptides (e.g., a full-length AspRS polypeptide and a truncatedAspRS polypeptide or two different truncated AspRS polypeptides), and/orheterodimers between an AspRS polypeptide and a heterologouspolypeptide. The monomers and/or dimmers may be soluble and may beisolated or purified to homogeneity. Certain heterodimers, such as thosebetween an AspRS polypeptide and a heterologous polypeptide, may bebi-functional.

Also included are monomers of AspRS polypeptides, including isolatedAspRS monomers that do not substantially dimerize with themselves(homodomerize) or with a second AspRS polypeptide (heterodimerize),whether due to one or more substitutions, truncations, deletions,additions, chemical modifications, or a combination of thesealterations. In certain embodiments, monomeric AspRS polypeptidespossess biological activities, including non-canonical activities, whichare not possessed by dimeric or multimeric AspRS polypeptide complexes.

In other embodiments, an AspRS polypeptide of the invention may be partof a multi-unit complex. A multi-unit complex of the present inventioncan include, for example, at least 2, 3, 4, or 5 or more monomers. Themonomers and/or multi-unit complexes of the present invention may besoluble and may be isolated or purified to homogeneity. Monomer units ofa multi-unit complex may be different, homologous, substantiallyhomologous, or identical to one another. However, a multi-unit complexof the invention includes at least one monomer comprising an AspRSpolypeptide as described herein or, in other embodiments, at least twoor more AspRS polypeptides as described herein.

Covalently linked monomers can be linked directly (by bonds) orindirectly (e.g., via a linker). For directly linking the polypeptidemonomers herein, it may be beneficial to modify the polypeptides hereinto enhance dimerization. For example, one or more amino acid residues ofan AspRS polypeptide may be modified by the addition or substation byone or more cysteines. Methods for creating amino acid substitutions,such as cysteine substitutions, or other modifications to facilitatelinking, are well known to those skilled in the art.

Certain embodiments of the present invention also contemplate the use ofmodified AspRS polypeptides, including modifications that improvedesired characteristics of an AspRS polypeptide, as described herein.Illustrative modifications of AspRS polypeptides of the inventioninclude, but are not limited to, chemical and/or enzymaticderivatizations at one or more constituent amino acids, including sidechain modifications, backbone modifications, and N- and C-terminalmodifications including acetylation, hydroxylation, methylation,amidation, and the attachment of carbohydrate or lipid moieties,cofactors, and the like. Exemplary modifications also include pegylationof an AspRS polypeptide (see, e.g., Veronese and Harris, Advanced DrugDelivery Reviews 54: 453-456, 2002, herein incorporated by reference).

In certain aspects, chemoselective ligation technology may be utilizedto modify truncated AspRS polypeptides of the invention, such as byattaching polymers in a site-specific and controlled manner. Suchtechnology typically relies on the incorporation of chemoselectiveanchors into the protein backbone by either chemical or recombinantmeans and subsequent modification with a polymer carrying acomplementary linker. As a result, the assembly process and the covalentstructure of the resulting protein-polymer conjugate may be controlled,enabling the rational optimization of drug properties, such as efficacyand pharmacokinetic properties (see, e.g., Kochendoerfer, CurrentOpinion in Chemical Biology 9:555-560, 2005).

The AspRS polypeptides described herein may be prepared by any suitableprocedure known to those of skill in the art, such as by recombinanttechniques. For example, AspRS polypeptides may be prepared by aprocedure including the steps of: (a) preparing a construct comprising apolynucleotide sequence that encodes an AspRS polypeptide and that isoperably linked to a regulatory element; (b) introducing the constructinto a host cell; (c) culturing the host cell to express the AspRSpolypeptide; and (d) isolating the AspRS polypeptide from the host cell.Recombinant AspRS polypeptides can be conveniently prepared usingstandard protocols as described for example in Sambrook, et al., (1989,supra), in particular Sections 16 and 17; Ausubel et al., (1994, supra),in particular Chapters 10 and 16; and Coligan et al., Current Protocolsin Protein Science (John Wiley & Sons, Inc. 1995-1997), in particularChapters 1, 5 and 6.

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc.85:2149-2154 (1963)). Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the desiredmolecule.

Polynucleotide Compositions

The present invention also provides isolated polynucleotides that encodethe AspRS polypeptides of the invention, as well as compositionscomprising such polynucleotides. Also included within the AspRSpolynucleotides of the present invention are primers, probes, antisenseoligonucleotides, and RNA interference agents that comprise all or aportion of the AspRS reference polynucleotides, which are complementaryto all or a portion of these reference polynucleotides, or whichspecifically hybridize to these reference polynucleotides, as describedherein.

As used herein, the terms “DNA” and “polynucleotide” and “nucleic acid”refer to a DNA molecule that has been isolated free of total genomic DNAof a particular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the polynucleotidesequences of this invention can include genomic sequences, extra-genomicand plasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an AspRS or a portion thereof) or may comprise avariant, or a biological functional equivalent of such a sequence.Polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions, as further described below,preferably such that the desired activity of the encoded polypeptide isnot substantially diminished relative to the unmodified polypeptide. Theeffect on the activity of the encoded polypeptide may generally beassessed as described herein.

In additional embodiments, the present invention provides isolatedpolynucleotides comprising various lengths of contiguous stretches ofsequence identical to or complementary to an aspartyl-tRNA synthetase,wherein the isolated polynucleotides encode an AspRS as describedherein. For example, polynucleotides are provided by this invention thatencode at least about 100, 150, 200, 250, 300, 350, 400, 450 or 500, ormore, contiguous amino acid residues of an AspRS polypeptide of theinvention, as well as all intermediate lengths. It will be readilyunderstood that “intermediate lengths”, in this context, means anylength between the quoted values, such as 101, 102, 103, etc.; 151, 152,153, etc.; 201, 202, 203, etc.

The polynucleotides of the present invention, regardless of the lengthof the coding sequence itself, may be combined with other DNA sequences,such as promoters, polyadenylation signals, additional restrictionenzyme sites, multiple cloning sites, other coding segments, and thelike, such that their overall length may vary considerably. It istherefore contemplated that a polynucleotide fragment of almost anylength may be employed; with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention, for example polynucleotides that are optimized forhuman and/or primate codon selection. Further, alleles of the genescomprising the polynucleotide sequences provided herein are within thescope of the present invention. Alleles are endogenous genes that arealtered as a result of one or more mutations, such as deletions,additions and/or substitutions of nucleotides. The resulting mRNA andprotein may, but need not, have an altered structure or function.Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

Polynucleotides and fusions thereof may be prepared, manipulated and/orexpressed using any of a variety of well established techniques knownand available in the art. For example, polynucleotide sequences whichencode polypeptides of the invention, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of an AspRS polypeptide in appropriate host cells. Due to theinherent degeneracy of the genetic code, other DNA sequences that encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and express agiven polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing,expression and/or activity of the gene product.

In order to express a desired polypeptide, a nucleotide sequenceencoding the polypeptide, or a functional equivalent, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,Current Protocols in Molecular Biology (1989).

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems, such as viral-based expression systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf. et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) geneswhich can be employed in tk- or aprt-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra).

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). These and otherassays are described, among other places, in Hampton et al., SerologicalMethods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med.158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins.

According to another aspect of the invention, polynucleotides encodingpolypeptides of the invention may be delivered to a subject in vivo,e.g., using gene therapy techniques. Gene therapy refers generally tothe transfer of heterologous nucleic acids to the certain cells, targetcells, of a mammal, particularly a human, with a disorder or conditionsfor which such therapy is sought. The nucleic acid is introduced intothe selected target cells in a manner such that the heterologous DNA isexpressed and a therapeutic product encoded thereby is produced.

Various viral vectors that can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, adeno-associatedvirus (AAV), or, preferably, an RNA virus such as a retrovirus.Preferably, the retroviral vector is a derivative of a murine or avianretrovirus, or is a lentiviral vector. The preferred retroviral vectoris a lentiviral vector. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a zinc finger derived-DNA binding polypeptidesequence of interest into the viral vector, along with another gene thatencodes the ligand for a receptor on a specific target cell, forexample, the vector may be made target specific. Retroviral vectors canbe made target specific by inserting, for example, a polynucleotideencoding a protein (dimer). Illustrative targeting may be accomplishedby using an antibody to target the retroviral vector. Those of skill inthe art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector containing the zinc finger-nucleotide binding proteinpolynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsulation. Helper cell lines which havedeletions of the packaging signal include but are not limited to .PSI.2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retroviral virions.

“Non-viral” delivery techniques for gene therapy can also be usedincluding, for example, DNA-ligand complexes, adenovirus-ligand-DNAcomplexes, direct injection of DNA, CaPO₄ precipitation, gene guntechniques, electroporation, liposomes, lipofection, and the like. Anyof these methods are widely available to one skilled in the art andwould be suitable for use in the present invention. Other suitablemethods are available to one skilled in the art, and it is to beunderstood that the present invention can be accomplished using any ofthe available methods of transfection. Lipofection can be accomplishedby encapsulating an isolated DNA molecule within a liposomal particleand contacting the liposomal particle with the cell membrane of thetarget cell. Liposomes are self-assembling, colloidal particles in whicha lipid bilayer, composed of amphiphilic molecules such as phosphatidylserine or phosphatidyl choline, encapsulates a portion of thesurrounding media such that the lipid bilayer surrounds a hydrophilicinterior. Unilammellar or multilammellar liposomes can be constructedsuch that the interior contains a desired chemical, drug, or, as in theinstant invention, an isolated DNA molecule.

Certain embodiments include polynucleotides that hybridize to areference AspRS polynucleotide sequence, or to their complements, understringency conditions described below. As used herein, the term“hybridizes under low stringency, medium stringency, high stringency, orvery high stringency conditions” describes conditions for hybridizationand washing. Guidance for performing hybridization reactions can befound in Ausubel et al., (1998, supra), Sections 6.3.1-6.3.6. Aqueousand non-aqueous methods are described in that reference and either canbe used.

Reference herein to low stringency conditions include and encompass fromat least about 1% v/v to at least about 15% v/v formamide and from atleast about 1 M to at least about 2 M salt for hybridization at 42° C.,and at least about 1 M to at least about 2 M salt for washing at 42° C.Low stringency conditions also may include 1% Bovine Serum Albumin(BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄(pH 7.2), 5% SDS for washing at room temperature. One embodiment of lowstringency conditions includes hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions).

Medium stringency conditions include and encompass from at least about16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9 M salt for hybridization at 42° C., and at leastabout 0.1 M to at least about 0.2 M salt for washing at 55° C. Mediumstringency conditions also may include 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and(i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2),5% SDS for washing at 60-65° C. One embodiment of medium stringencyconditions includes hybridizing in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 60° C. High stringencyconditions include and encompass from at least about 31% v/v to at leastabout 50% v/v formamide and from about 0.01 M to about 0.15 M salt forhybridization at 42° C., and about 0.01 M to about 0.02 M salt forwashing at 55° C.

High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 MNaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC,0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS forwashing at a temperature in excess of 65° C. One embodiment of highstringency conditions includes hybridizing in 6×SSC at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. Oneembodiment of very high stringency conditions includes hybridizing in0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washesin 0.2×SSC, 1% SDS at 65° C.

Other stringency conditions are well known in the art and a skilledartisan will recognize that various factors can be manipulated tooptimize the specificity of the hybridization. Optimization of thestringency of the final washes can serve to ensure a high degree ofhybridization. For detailed examples, see Ausubel et al., supra at pages2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to1.104.

While stringent washes are typically carried out at temperatures fromabout 42° C. to 68° C., one skilled in the art will appreciate thatother temperatures may be suitable for stringent conditions. Maximumhybridization rate typically occurs at about 20° C. to 25° C. below theT_(m) for formation of a DNA-DNA hybrid. It is well known in the artthat the T_(m) is the melting temperature, or temperature at which twocomplementary polynucleotide sequences dissociate. Methods forestimating T_(m) are well known in the art (see Ausubel et al., supra atpage 2.10.8).

In general, the T_(m) of a perfectly matched duplex of DNA may bepredicted as an approximation by the formula: T_(m)=81.5+16.6 (log₁₀M)+0.41 (% G+C)−0.63 (% formamide)−(600/length) wherein: M is theconcentration of Na⁺, preferably in the range of 0.01 molar to 0.4molar; % G+C is the sum of guanosine and cytosine bases as a percentageof the total number of bases, within the range between 30% and 75% G+C;% formamide is the percent formamide concentration by volume; length isthe number of base pairs in the DNA duplex. The T_(m) of a duplex DNAdecreases by approximately 1° C. with every increase of 1% in the numberof randomly mismatched base pairs. Washing is generally carried out atT_(m)−15° C. for high stringency, or T_(m)−30° C. for moderatestringency.

In one example of a hybridization procedure, a membrane (e.g., anitrocellulose membrane or a nylon membrane) containing immobilized DNAis hybridized overnight at 42° C. in a hybridization buffer (50%deionized formamide, 5×SSC, 5×Denhardt's solution (0.1% ficoll, 0.1%polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200mg/mL denatured salmon sperm DNA) containing a labeled probe. Themembrane is then subjected to two sequential medium stringency washes(i.e., 2×SSC, 0.1% SDS for 15 min at 45° C., followed by 2×SSC, 0.1% SDSfor 15 min at 50° C.), followed by two sequential higher stringencywashes (i.e., 0.2×SSC, 0.1% SDS for 12 min at 55° C. followed by 0.2×SSCand 0.1% SDS solution for 12 min at 65-68° C.

Embodiments of the present invention also include oligonucleotides,whether for detection, amplification, antisense therapies, or otherpurpose. For these and related purposes, the term “oligonucleotide” or“oligo” or “oligomer” is intended to encompass a singular“oligonucleotide” as well as plural “oligonucleotides,” and refers toany polymer of two or more of nucleotides, nucleosides, nucleobases orrelated compounds used as a reagent in the amplification methods of thepresent invention, as well as subsequent detection methods. Theoligonucleotide may be DNA and/or RNA and/or analogs thereof.

The term oligonucleotide does not necessarily denote any particularfunction to the reagent, rather, it is used generically to cover allsuch reagents described herein. An oligonucleotide may serve variousdifferent functions, e.g., it may function as a primer if it is capableof hybridizing to a complementary strand and can further be extended inthe presence of a nucleic acid polymerase, it may provide a promoter ifit contains a sequence recognized by an RNA polymerase and allows fortranscription, and it may function to prevent hybridization or impedeprimer extension if appropriately situated and/or modified. Anoligonucleotide may also function as a probe, or an antisense agent. Anoligonucleotide can be virtually any length, limited only by itsspecific function, e.g., in an amplification reaction, in detecting anamplification product of the amplification reaction, or in an antisenseor RNA interference application. Any of the oligonucleotides describedherein can be used as a primer, a probe, an antisense oligomer, or anRNA interference agent.

The term “primer” as used herein refers to a single-strandedoligonucleotide capable of acting as a point of initiation fortemplate-directed DNA synthesis under suitable conditions defined, forexample, by buffer and temperature, in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as a DNAor RNA polymerase or reverse transcriptase. The length of the primer, inany given case, depends on, for example, the intended use of the primer,and generally ranges from about 15 to 30 nucleotides, although shorterand longer primers may be used. Short primer molecules generally requirecooler temperatures to form sufficiently stable hybrid complexes withthe template. A primer need not reflect the exact sequence of thetemplate but must be sufficiently complementary to hybridize with suchtemplate. The primer site is the area of the template to which a primerhybridizes. The primer pair is a set of primers including a 5′ upstreamprimer that hybridizes with the 5′ end of the sequence to be amplifiedand a 3′ downstream primer that hybridizes with the complement of the 3′end of the sequence to be amplified.

The term “probe” as used herein refers to a surface-immobilized moleculethat can be recognized by a particular target. See, e.g., U.S. Pat. No.6,582,908 for an example of arrays having all possible combinations ofprobes with 10, 12, and more bases. Probes and primers as used hereintypically comprise at least 10-15 contiguous nucleotides of a knownsequence. In order to enhance specificity, longer probes and primers mayalso be employed, such as probes and primers that comprise at least 20,25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 nucleotides of anAspRS reference sequence or its complement. Probes and primers may beconsiderably longer than these examples, and it is understood that anylength supported by the knowledge in the art and the specification,including the tables, figures, and Sequence Listing, may be used.

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook, J. et al. (1989) Molecular Cloning: ALaboratory Manual, 2.sup.nd ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; Ausubel, F. M. et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New YorkN.Y.; Innis, M. et al. (1990) PCR Protocols. A Guide to Methods andApplications, Academic Press, San Diego Calif. PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers or probes may be selected usingsoftware known in the art. For example, OLIGO 4.06 software is usefulfor the selection of PCR primer pairs of up to 100 nucleotides each, andfor the analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. The Primer3 primer selection program (available to the publicfrom the Whitehead Institute/MIT Center for Genome Research, CambridgeMass.) allows the user to input a “mispriming library,” in whichsequences to avoid as primer binding sites are user-specified. Theoligonucleotides and polynucleotide fragments identified by any of theabove selection methods are useful in hybridization technologies, forexample, as PCR or sequencing primers, microarray elements, or specificprobes to identify fully or partially complementary polynucleotides in asample of nucleic acids. Methods of oligonucleotide selection are notlimited to those described herein.

The terms “antisense oligomer” or “antisense compound” or “antisenseoligonucleotide” are used interchangeably and refer to a sequence ofcyclic subunits, each bearing a base-pairing moiety, linked byintersubunit linkages that allow the base-pairing moieties to hybridizeto a target sequence in a nucleic acid (typically an RNA) byWatson-Crick base pairing, to form a nucleic acid:oligomer heteroduplexwithin the target sequence, and typically thereby prevent translation ofthat RNA. Also included are methods of use thereof to modulateexpression of a selected AspRS transcript, such as a splice variant orproteolytic fragment, and/or its corresponding polyeptide.

Antisense oligonucleotides may contain between about 8 and 40 subunits,typically about 8-25 subunits, and preferably about 12 to 25 subunits.In certain embodiments, oligonucleotides may have exact sequencecomplementarity to the target sequence or near complementarity, asdefined below. In certain embodiments, the degree of complementaritybetween the target and antisense targeting sequence is sufficient toform a stable duplex. The region of complementarity of the antisenseoligomers with the target RNA sequence may be as short as 8-11 bases,but is preferably 12-15 bases or more, e.g., 12-20 bases, or 12-25bases, including all integers in between these ranges. An antisenseoligomer of about 14-15 bases is generally long enough to have a uniquecomplementary sequence in targeting the selected AspRS transcript.

In certain embodiments, antisense oligomers as long as 40 bases may besuitable, where at least a minimum number of bases, e.g., 10-12 bases,are complementary to the target sequence. In general, however,facilitated or active uptake in cells is optimized at oligomer lengthsless than about 30. For certain oligomers, described further below, anoptimum balance of binding stability and uptake generally occurs atlengths of 18-25 bases. Included are antisense oligomers (e.g., PNAs,LNAs, 2′-OMe, MOE, morpholinos) that consist of about 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40 bases, in which at least about 6,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguousor non-contiguous bases are complementary to their AspRS targetsequence, or variants thereof.

In certain embodiments, antisense oligomers may be 100% complementary tothe AspRS nucleic acid target sequence, or it may include mismatches,e.g., to accommodate variants, as long as a heteroduplex formed betweenthe oligomer and AspRS nucleic acid target sequence is sufficientlystable to withstand the action of cellular nucleases and other modes ofdegradation which may occur in vivo. Oligomer backbones which are lesssusceptible to cleavage by nucleases are discussed below. Mismatches, ifpresent, are less destabilizing toward the end regions of the hybridduplex than in the middle. The number of mismatches allowed will dependon the length of the oligomer, the percentage of G:C base pairs in theduplex, and the position of the mismatch(es) in the duplex, according towell understood principles of duplex stability. Although such anantisense oligomer is not necessarily 100% complementary to the AspRSnucleic acid target sequence, it is effective to stably and specificallybind to the target sequence, such that a biological activity of thenucleic acid target, e.g., expression of AspRS protein(s), is modulated.

The stability of the duplex formed between an oligomer and a targetsequence is a function of the binding Tm and the susceptibility of theduplex to cellular enzymatic cleavage. The Tm of an antisenseoligonucleotide with respect to complementary-sequence RNA may bemeasured by conventional methods, such as those described by Hames etal., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or asdescribed in Miyada C. G. and Wallace R. B., 1987, Oligonucleotidehybridization techniques, Methods Enzymol. Vol. 154 pp. 94-107. Incertain embodiments, antisense oligomer may have a binding Tm, withrespect to a complementary-sequence RNA, of greater than bodytemperature and preferably greater than 50° C. Tm's in the range 60-80°C. or greater are preferred. According to well known principles, the Tmof an oligomer compound, with respect to a complementary-based RNAhybrid, can be increased by increasing the ratio of C:G paired bases inthe duplex, and/or by increasing the length (in base pairs) of theheteroduplex.

Antisense oligomers can be designed to block or inhibit translation ofmRNA or to inhibit natural pre-mRNA splice processing, or inducedegradation of targeted mRNAs, and may be said to be “directed to” or“targeted against” a target sequence with which it hybridizes. Incertain embodiments, the target sequence may include any coding ornon-coding sequence of an AspRS mRNA transcript, and may thus by withinan exon or within an intron. In certain embodiments, the target sequenceis relatively unique or exceptional among AspRS s and is selective forreducing expression of a selected AspRS proteolytic fragment or splicevariant. In certain embodiments, the target site includes a 3′ or 5′splice site of a pre-processed mRNA, or a branch point. The targetsequence for a splice site may include an mRNA sequence having its 5′end 1 to about 25 to about 50 base pairs downstream of a splice acceptorjunction or upstream of a splice donor junction in a preprocessed mRNA.An oligomer is more generally said to be “targeted against” abiologically relevant target, such as reference AspRS polynucleotide,when it is targeted against the nucleic acid of the target in the mannerdescribed herein.

A “subunit” of an oligonucleotide refers to one nucleotide (ornucleotide analog) unit. The term may refer to the nucleotide unit withor without the attached intersubunit linkage, although, when referringto a “charged subunit”, the charge typically resides within theintersubunit linkage (e.g., a phosphate or phosphorothioate linkage or acationic linkage).

The cyclic subunits of an oligonucleotide may be based on ribose oranother pentose sugar or, in certain embodiments, alternate or modifiedgroups. Examples of modified oligonucleotide backbones include, withoutlimitation, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Also contemplated are peptide nucleic acids(PNAs), locked nucleic acids (LNAs), 2′-O-Methyl oligonucleotides(2′-OMe), 2′-methoxyethoxy oligonucleotides (MOE), morpholinos, amongother oligonucleotides known in the art.

The purine or pyrimidine base pairing moiety is typically adenine,cytosine, guanine, uracil, thymine or inosine. Also included are basessuch as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,2,4,6-trime115thoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl,aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines(e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne,quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, β-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonyhnethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleotide bases other than adenine (A), guanine (G), cytosine(C), thymine (T), and uracil (U), as illustrated above; such bases canbe used at any position in the antisense molecule. Persons skilled inthe art will appreciate that depending on the uses of the oligomers, Tsand Us are interchangeable. For instance, with other antisensechemistries such as 2′-O-methyl antisense oligonucleotides that are moreRNA-like, the T bases may be shown as U.

An oligonucleotide is typically complementary to a target sequence, suchas a target DNA or RNA. The terms “complementary” and “complementarity”refer to polynucleotides (i.e., a sequence of nucleotides) related bythe base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity (100%) between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. While perfect complementarity is often desired, someembodiments can include one or more but preferably 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches withrespect to the target sequence. Variations at any location within theoligomer are included. In certain embodiments, variations in sequencenear the termini of an oligomer are generally preferable to variationsin the interior, and if present are typically within about 10, 9, 8, 7,6, 5, 4, 3, 2, or 1 nucleotides of the 5′ and/or 3′ terminus.

The term “target sequence” refers to a portion of the target RNA againstwhich the oligonucleotide is directed, that is, the sequence to whichthe oligonucleotide will hybridize by Watson-Crick base pairing of acomplementary sequence. In certain embodiments, the target sequence maybe a contiguous region of an AspRS mRNA (e.g., a unique splice junctionof an AspRS mRNA), or may be composed of non-contiguous regions of themRNA.

The term “targeting sequence” or in certain embodiments “antisensetargeting sequence” refers to the sequence in an oligonucleotide that iscomplementary (meaning, in addition, substantially complementary) to thetarget sequence in the DNA or RNA target molecule. The entire sequence,or only a portion, of the antisense compound may be complementary to thetarget sequence. For example, in an oligonucleotide having 20-30 bases,about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, or 29 may be targeting sequences that arecomplementary to the target region. Typically, the targeting sequence isformed of contiguous bases, but may alternatively be formed ofnon-contiguous sequences that when placed together, e.g., from oppositeends of the oligonucleotide, constitute sequence that spans the targetsequence.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, it may still be functionally “complementary.”

An oligonucleotide “specifically hybridizes” to a target polynucleotideif the oligomer hybridizes to a target (e.g., an AspRS referencepolynucleotide or its complement) under physiological conditions, with aTm substantially greater than 45° C., preferably at least 50° C., andtypically 60° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions. At a given ionicstrength and pH, the Tm is the temperature at which 50% of a targetsequence hybridizes to a complementary polynucleotide. Again, suchhybridization may occur with “near” or “substantial” complementarity ofthe antisense oligomer to the target sequence, as well as with exactcomplementarity.

The terms specifically binds or specifically hybridizes refer generallyto an oligonucleotide probe or polynucleotide sequence that not onlybinds to its intended target gene sequence in a sample under selectedhybridization conditions, but does not bind significantly to othertarget sequences in the sample, and thereby discriminates between itsintended target and all other targets in the target pool. A probe thatspecifically hybridizes to its intended target sequence may also detectconcentration differences under the selected hybridization conditions,as described herein.

As noted above, certain oligonucleotides provided herein include peptidenucleic acids (PNAs). Also included are “locked nucleic acid” subunits(LNAs). The structures of LNAs are known in the art: for example,Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998)54, 3607, and Accounts of Chem. Research (1999) 32, 301); Obika, et al.,Tetrahedron Letters (1997) 38, 8735; (1998) 39, 5401, and BioorganicMedicinal Chemistry (2008)16, 9230. Certain oligonucleotides maycomprise morpholino-based subunits bearing base-pairing moieties, joinedby uncharged or substantially uncharged linkages. The terms “morpholinooligomer” or “PMO” (phosphoramidate- or phosphorodiamidate morpholinooligomer) refer to an oligonucleotide analog composed of morpholinosubunit structures, where (i) the structures are linked together byphosphorus-containing linkages, one to three atoms long, preferably twoatoms long, and preferably uncharged or cationic, joining the morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit,and (ii) each morpholino ring bears a purine or pyrimidine or anequivalent base-pairing moiety effective to bind, by base specifichydrogen bonding, to a base in a polynucleotide.

In certain embodiments, oligonucleotides can be prepared by stepwisesolid-phase synthesis, employing methods detailed in the referencescited above, and below with respect to the synthesis of oligonucleotideshaving a mixture or uncharged and cationic backbone linkages. In somecases, it may be desirable to add additional chemical moieties to theoligonucleotide, e.g., to enhance pharmacokinetics or to facilitatecapture or detection of the compound. Such a moiety may be covalentlyattached, typically to a terminus of the oligomer, according to standardsynthetic methods. For example, addition of a polyethylene glycol moietyor other hydrophilic polymer, e.g., one having 10-100 monomericsubunits, may be useful in enhancing solubility. One or more chargedgroups, e.g., anionic charged groups such as an organic acid, mayenhance cell uptake.

A variety of detectable molecules may be used to render anoligonucleotide detectable, such as a radioisotopes, fluorochromes,dyes, enzymes, nanoparticles, chemiluminescent markers, biotin, or othermonomer known in the art that can be detected directly (e.g., by lightemission) or indirectly (e.g., by binding of a fluorescently-labeledantibody).

Certain embodiments relate to RNA interference (RNAi) agents that targetone or more mRNA transcripts of an AspRS reference polynucleotide,including fragments and variants thereof. Also included are methods ofuse thereof to modulate the levels of a selected AspRS transcript, suchas an AspRS splice variant or proteolytic fragment.

The term “double-stranded” means two separate nucleic acid strandscomprising a region in which at least a portion of the strands aresufficiently complementary to hydrogen bond and form a duplex structure.The term “duplex” or “duplex structure” refers to the region of a doublestranded molecule wherein the two separate strands are substantiallycomplementary, and thus hybridize to each other. “dsRNA” refers to aribonucleic acid molecule having a duplex structure comprising twocomplementary and anti-parallel nucleic acid strands (i.e., the senseand antisense strands). Not all nucleotides of a dsRNA must exhibitWatson-Crick base pairs; the two RNA strands may be substantiallycomplementary. The RNA strands may have the same or a different numberof nucleotides.

The strands of a dsRNA are sufficiently complementary to hybridize toform a duplex structure. In certain embodiments, the complementary RNAstrand may be less than 30 nucleotides, less than 25 nucleotides inlength, or even 19 to 24 nucleotides in length. In certain aspects, thecomplementary nucleotide sequence may be 20-23 nucleotides in length, or22 nucleotides in length.

In certain embodiments, at least one of the RNA strands comprises anucleotide overhang of 1 to 4 nucleotides in length. In otherembodiments, one or both of the strands are blunt-ended. In certainembodiments, the dsRNA may further comprise at least one chemicallymodified nucleotide.

Certain embodiments of the present invention may comprise microRNAs.Micro-RNAs represent a large group of small RNAs produced naturally inorganisms, some of which regulate the expression of target genes.Micro-RNAs are formed from an approximately 70 nucleotidesingle-stranded hairpin precursor transcript by Dicer. (V. Ambros et al.Current Biology 13:807, 2003).

Certain embodiments may also employ short-interfering RNAs (siRNA). Eachstrand of an siRNA agent can be equal to or less than 35, 30, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length. The strandis preferably at least 19 nucleotides in length. For example, eachstrand can be between 21 and 25 nucleotides in length. Preferred siRNAagents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25nucleotide pairs, and one or more overhangs, preferably one or two 3′overhangs, of 2-3 nucleotides.

A “single strand RNAi agent” as used herein, is an RNAi agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. A single strand RNAi agent is at least 14, andmore preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides inlength. It is preferably less than 200, 100, or 60 nucleotides inlength.

Hairpin RNAi modulating agents may have a duplex region equal to or atleast 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplexregion may preferably be equal to or less than 200, 100, or 50, inlength. Certain ranges for the duplex region are 15-30, 17 to 23, 19 to23, and 19 to 21 nucleotides pairs in length. The hairpin may have asingle strand overhang or terminal unpaired region, preferably the 3′,and preferably of the antisense side of the hairpin. In certainembodiments, overhangs are 2-3 nucleotides in length.

The present invention further encompasses oligonucleotides employingribozymes. Also included are vector delivery systems that are capable ofexpressing the AspRS-targeting sequences described herein. Included arevectors that express siRNA or other duplex-forming RNA interferencemolecules. Exemplary delivery systems may include viral vector systems(i.e., viral-mediated transduction) including, but not limited to,retroviral (e.g., lentiviral) vectors, adenoviral vectors,adeno-associated viral vectors, and herpes viral vectors, among othersknown in the art.

Oligonucleotides and RNAi agents that target one or more portions of anAspRS polynucleotide reference sequence or its complement may be used inany of the therapeutic, diagnostic, or drug screening methods describedherein and apparent to persons skilled in the art.

Antibody Compositions, Fragments Thereof and Other Binding Agents

According to another aspect, the present invention further providesbinding agents, such as antibodies, antigen-binding fragments thereof,soluble receptors, small molecules, aptamers etc., that exhibit bindingspecificity for a polypeptide disclosed herein, or to a portion, variantor derivative thereof, and methods of using same. Preferably, suchbinding agents are effective for modulating one or more of thenon-canonical activities mediated by an AspRS polypeptide of theinvention. In certain embodiments, for example, the binding agent is onethat binds to an AspRS polypeptide of the invention and inhibits itsability to bind to one or more of its cellular binding partners.Accordingly, such binding agents may be used to treat or preventdiseases, disorders or other conditions that are mediated by an AspRSpolypeptide of the invention by antagonizing it activity.

An antibody, or antigen-binding fragment thereof, is said to“specifically bind,” “immunologically bind,” and/or is “immunologicallyreactive” to a polypeptide of the invention if it reacts at a detectablelevel (within, for example, an ELISA assay) with the polypeptide, anddoes not react detectably with unrelated polypeptides under similarconditions.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, e.g., Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

An “antigen-binding site,” or “binding portion” of an antibody, refersto the part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs”. Thus the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

A binding agent may be, for example, a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. Monoclonal antibodies specific for a polypeptide ofinterest may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.The polypeptides of this invention may be used in the purificationprocess in, for example, an affinity chromatography step.

An “Fv” fragment can be produced by preferential proteolytic cleavage ofan IgM, and on rare occasions IgG or IgA immunoglobulin molecule. Fvfragments are, however, more commonly derived using recombinanttechniques known in the art. The Fv fragment includes a non-covalentV_(H)::V_(L) heterodimer including an antigen-binding site which retainsmuch of the antigen recognition and binding capabilities of the nativeantibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich etal. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRS and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRS. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

As noted above, “peptides” are included as binding agents. The termpeptide typically refers to a polymer of amino acid residues and tovariants and synthetic analogues of the same. In certain embodiments,the term “peptide” refers to relatively short polypeptides, includingpeptides that consist of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids,including all integers and ranges (e.g., 5-10, 8-12, 10-15) in between,and interact with an AspRS polypeptide, its cellular binding partner, orboth. Peptides can be composed of naturally-occurring amino acids and/ornon-naturally occurring amino acids, as described herein.

A binding agent may include a peptide mimetic or other small molecule. A“small molecule” refers to an organic compound that is of synthetic orbiological origin (biomolecule), but is typically not a polymer. Organiccompounds refer to a large class of chemical compounds whose moleculescontain carbon, typically excluding those that contain only carbonates,simple oxides of carbon, or cyanides. A “biomolecule” refers generallyto an organic molecule that is produced by a living organism, includinglarge polymeric molecules (biopolymers) such as peptides,polysaccharides, and nucleic acids as well, and small molecules such asprimary secondary metabolites, lipids, phospholipids, glycolipids,sterols, glycerolipids, vitamins, and hormones. A “polymer” refersgenerally to a large molecule or macromolecule composed of repeatingstructural units, which are typically connected by covalent chemicalbond.

In certain embodiments, a small molecule has a molecular weight of lessthan 1000 Daltons, typically between about 300 and 700 Daltons, andincluding about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,500, 650, 600, 750, 700, 850, 800, 950, or 1000 Daltons.

Aptamers are also included as binding agents. Examples of aptamersincluded nucleic acid aptamers (e.g., DNA aptamers, RNA aptamers) andpeptide aptamers. Nucleic acid aptamers refer generally to nucleic acidspecies that have been engineered through repeated rounds of in vitroselection or equivalent method, such as SELEX (systematic evolution ofligands by exponential enrichment), to bind to various molecular targetssuch as small molecules, proteins, nucleic acids, and even cells,tissues and organisms. Hence, included are nucleic acid aptamers thatbind to the AspRS polypeptides described herein and/or their cellularbinding partners.

Peptide aptamers typically include a variable peptide loop attached atboth ends to a protein scaffold, a double structural constraint thattypically increases the binding affinity of the peptide aptamer tolevels comparable to that of an antibody's (e.g., in the nanomolarrange). In certain embodiments, the variable loop length may be composedof about 10-20 amino acids (including all integers in between), and thescaffold may include any protein that has good solubility and compacityproperties. Certain exemplary embodiments may utilize the bacterialprotein Thioredoxin-A as a scaffold protein, the variable loop beinginserted within the reducing active site (-Cys-Gly-Pro-Cys- loop in thewild protein), with the two cysteines lateral chains being able to forma disulfide bridge. Hence, included are peptide aptamers that bind tothe AspRS polypeptides described herein and/or their cellular bindingpartners. Peptide aptamer selection can be performed using differentsystems known in the art, including the yeast two-hybrid system.

As noted above, the AspRS polypeptides and binding agents of the presentinvention can be used in any of the diagnostic, drug discovery, ortherapeutic methods described herein.

In another embodiment of the invention, binding agents such asmonoclonal antibodies of the present invention may be coupled to one ormore agents of interest. For example, a therapeutic agent may be coupled(e.g., covalently bonded) to a suitable monoclonal antibody eitherdirectly or indirectly (e.g., via a linker group). A direct reactionbetween an agent and an antibody is possible when each possesses asubstituent capable of reacting with the other. For example, anucleophilic group, such as an amino or sulfhydryl group, on one may becapable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used.

Formulation and Administration

The compositions of the invention (e.g., polypeptides, polynucleotides,antibodies, etc.) are generally formulated inpharmaceutically-acceptable or physiologically-acceptable solutions foradministration to a cell, tissue or animal, either alone, or incombination with one or more other modalities of therapy. It will alsobe understood that, if desired, the compositions of the invention may beadministered in combination with other agents as well, such as, e.g.,other proteins or polypeptides or various pharmaceutically-activeagents. There is virtually no limit to other components that may also beincluded in the compositions, provided that the additional agents do notadversely affect the desired effects desired to be achieved with anAspRS polypeptide of the invention.

In the pharmaceutical compositions of the invention, formulation ofpharmaceutically-acceptable excipients and carrier solutions iswell-known to those of skill in the art, as is the development ofsuitable dosing and treatment regimens for using the particularcompositions described herein in a variety of treatment regimens,including e.g., oral, parenteral, intravenous, intranasal, intracranialand intramuscular administration and formulation.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to a subject. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as described,for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 andU.S. Pat. No. 5,399,363 (each specifically incorporated herein byreference in its entirety). Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form should be sterileand should be fluid to the extent that easy syringability exists. Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with thevarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, polynucleotides, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212(each specifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticleor the like. The formulation and use of such delivery vehicles can becarried out using known and conventional techniques.

Kits Comprising Compositions of the Invention

The invention, in other aspects, provides kits comprising one or morecontainers filled with one or more of the polypeptides, polynucleotides,antibodies, multiunit complexes, compositions thereof, etc., of theinvention, as described herein. The kits can include writteninstructions on how to use such compositions (e.g., to modulate cellularsignaling, angiogenesis, cancer, inflammatory conditions, etc.).

The kits herein may also include a one or more additional therapeuticagents or other components suitable or desired for the indication beingtreated. An additional therapeutic agent may be contained in a secondcontainer, if desired. Examples of additional therapeutic agentsinclude, but are not limited to antineoplastic agents, anti-inflammatoryagents, antibacterial agents, antiviral agents, angiogenic agents, etc.

The kits herein can also include one or more syringes or othercomponents necessary or desired to facilitate an intended mode ofdelivery (e.g., stents, implantable depots, etc.).

Methods of Use

Embodiments of the present invention also include methods of using theAspRS compositions or “agents” described herein for diagnostic, drugdiscovery, and/or therapeutic purposes. The term AspRS “agents” refersgenerally to the AspRS polynucleotides, AspRS polypeptides, bindingagents, and other compounds described herein. For diagnostic purposes,the AspRS agents can be used in a variety of non-limiting ways, such asto distinguish between different cell types or different cellularstates, or to identify subjects having a relevant disease or condition.For drug discovery purposes, the AspRS agents can be used to identifyone or more cellular “binding partners” of an AspRS polypeptide,characterize one or more “non-canonical” activities of an AspRSpolypeptide, identify agents that selectively or non-selectively agonizeor antagonize the interaction of an AspRS polypeptide with its bindingpartner(s), and/or identify agents that selectively or non-selectivelyagonize or antagonize one or more “non-canonical” activities of an AspRSpolypeptide. For therapeutic purposes, the AspRS agents or compositionsprovided herein can be used to treat a variety of diseases orconditions, detailed below.

A. Diagnostics

As noted above, AspRS agents described herein can be used in diagnosticassays. These embodiments include the detection of the AspRSpolynucleotide sequence(s) or corresponding polypeptide sequence(s) orportions thereof of one or more newly identified AspRS proteinfragments. In certain embodiments, the presence or levels of one or morenewly identified AspRS sequences associates or correlates with one ormore cellular types or cellular states. Hence, as noted above, thepresence or levels of an AspRS sequence can be used to distinguishbetween different cellular types or different cellular states. Thepresence or levels of AspRS sequences can be detected according topolynucleotide and/or polypeptide-based diagnostic techniques.

Certain of the methods provided herein rely on the differentialexpression of an AspRS sequence to characterize the condition or stateof a cell, tissue, or subject, and to distinguish it from another cell,tissue, or subject. Non-limiting examples include methods of detectingthe presence or levels of an AspRS sequence in a biological sample todistinguish between cells or tissues of different species, cells ofdifferent tissues or organs, cellular developmental states such asneonatal and adult, cellular differentiation states, conditions such ashealthy, diseased and treated, intracellular and extracellularfractions, in addition to primary cell cultures and other cell cultures,such as immortalized cell cultures.

Differential expression refers generally to a statistically significantdifference in one or more gene expression levels of an AspRSpolynucleotide or polypeptide sequence compared to the expression levelsof the same sequence in an appropriate control. The statisticallysignificant difference may relate to either an increase or a decrease inexpression levels, as measured by RNA levels, protein levels, proteinfunction, or any other relevant measure of gene expression such as thosedescribed herein.

A result is typically referred to as statistically significant if it isunlikely to have occurred by chance. The significance level of a test orresult relates traditionally to a frequentist statistical hypothesistesting concept. In simple cases, statistical significance may bedefined as the probability of making a decision to reject the nullhypothesis when the null hypothesis is actually true (a decision knownas a Type I error, or “false positive determination”). This decision isoften made using the p-value: if the p-value is less than thesignificance level, then the null hypothesis is rejected. The smallerthe p-value, the more significant the result. Bayes factors may also beutilized to determine statistical significance (see, e.g., Goodman S.,Ann Intern Med 130:1005-13, 1999).

In more complicated, but practically important cases, the significancelevel of a test or result may reflect an analysis in which theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true is no more than the stated probability.This type of analysis allows for those applications in which theprobability of deciding to reject may be much smaller than thesignificance level for some sets of assumptions encompassed within thenull hypothesis.

In certain exemplary embodiments, statistically significant differentialexpression may include situations wherein the expression level of agiven AspRS sequence provides at least about a 1.2×, 1.3×, 1.4×, 1.5×,1.6×, 1.7×, 1.8×, 1.9×. 2.0×, 2.2×, 2.4×, 2.6×, 2.8×, 3.0×, 4.0×, 5.0×,6.0×, 7.0×, 8.0×, 9.0×, 10.0×, 15.0×, 20.0×, 50.0×, 100.0×, or greaterdifference in expression (i.e., differential expression that may behigher or lower expression) in a suspected biological sample as comparedto an appropriate control, including all integers and decimal points inbetween (e.g., 1.24×, 1.25×, 2.1×, 2.5×, 60.0×, 75.0×, etc.). In certainembodiments, statistically significant differential expression mayinclude situations wherein the expression level of a given AspRSsequence provides at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 percent (%) or greaterdifference in expression (i.e., differential expression that may behigher or lower) in a suspected biological sample as compared to anappropriate control, including all integers and decimal points inbetween.

As an additional example, differential expression may also be determinedby performing Z-testing, i.e., calculating an absolute Z score, asdescribed herein and known in the art (see Example 1). Z-testing istypically utilized to identify significant differences between a samplemean and a population mean. For example, as compared to a standardnormal table (e.g., a control tissue), at a 95% confidence interval(i.e., at the 5% significance level), a Z-score with an absolute valuegreater than 1.96 indicates non-randomness. For a 99% confidenceinterval, if the absolute Z is greater than 2.58, it means that p<0.01,and the difference is even more significant—the null hypothesis can berejected with greater confidence. In these and related embodiments, anabsolute Z-score of 1.96, 2, 2.58, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more, including all decimal points inbetween (e.g., 10.1, 10.6, 11.2, etc.), may provide a strong measure ofstatistical significance. In certain embodiments, an absolute Z-score ofgreater than 6 may provide exceptionally high statistical significance.

Substantial similarly relates generally to the lack of a statisticallysignificant difference in the expression levels between the biologicalsample and the reference control. Examples of substantially similarexpression levels may include situations wherein the expression level ofa given SSCIGS provides less than about a 0.05×, 0.1×, 0.2×, 0.3×, 0.4×,0.5×, 0.6×, 0.7×, 0.8×, 0.9×. 1.0×, 1.1×, 1.2×, 1.3×, or 1.4× differencein expression (i.e., differential expression that may be higher or lowerexpression) in a suspected biological sample as compared to a referencesample, including all decimal points in between (e.g., 0.15×, 0.25×,0.35×, etc.). In certain embodiments, differential expression mayinclude situations wherein the expression level of a given AspRSsequence provides less than about 0.25. 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 percent (%)difference in expression (i.e., differential expression that may behigher or lower) in a suspected biological sample as compared to areference sample, including all decimal points in between.

In certain embodiments, such as when using an Affymetrix Microarray tomeasure the expression levels of an AspRS polynucleotide or polypeptidesequence, differential expression may also be determined by the meanexpression value summarized by Affymetrix Microarray Suite 5 software(Affymetrix, Santa Clara, Calif.), or other similar software, typicallywith a scaled mean expression value of 1000.

Embodiments of the present invention include methods of detecting thepresence or levels of an AspRS polynucleotide or polypeptide referencesequence or a portion thereof to distinguish between cells, tissues, orother biological samples of a different organism or species, wherein thepresence or levels of that sequence associates with a selected organismor species. General examples include methods of distinguishing betweenhumans and any combination of bacteria, fungi, plants, and othernon-human animals. Included within animals are methods of distinguishingbetween humans and any combination of vertebrates and invertebrates,including vertebrates such as fish, amphibians, reptiles, birds, andnon-human mammals, and inverterbrates such as insects, mollusks,crustaceans, and corals. Included within non-human mammals are methodsof distinguishing between humans and any combination of non-humanmammals from the Order Afrosoricida, Macroscelidea, Tubulidentata,Hyracoidea, Proboscidea, Sirenia, Cingulata, Pilosa, Scandentia,Dermoptera, Primates, Rodentia, Lagomorpha, Erinaceomorpha,Soricomorpha, Chiroptera, Pholidota, Cetacea, Carnivora, Perissodactyla,or Artiodactyla. Included within the Primate Order are monkeys, apes,gorillas, and chimpanzees, among others known in the art. Accordingly,the presence or levels of an AspRS polynucleotide or polypeptidereference sequence or variant, as described herein, may be used toidentify the source of a given biological sample, such as a cell,tissue, or organ, by distinguishing between any combination of theseorganisms, or by distinguishing between humans and any one or more ofthese organisms, such as a panel of organisms. In certain embodiments,the source of a given biological sample may also be determined bycomparing the presence or levels of an AspRS sequence or a portionthereof to a pre-determined value.

Embodiments of the present invention include methods of detecting thepresence or levels of an AspRS polynucleotide or polypeptide referencesequence or a portion thereof to distinguish between cells or otherbiological samples that originate from different tissues or organs.Non-limiting examples include methods of distinguishing between a cellor other biological sample that originates from any combination of skin(e.g., dermis, epidermis, subcutaneous layer), hair follicles, nervoussystem (e.g., brain, spinal cord, peripheral nerves), auditory system orbalance organs (e.g., inner ear, middle ear, outer ear), respiratorysystem (e.g., nose, trachea, lungs), gastroesophogeal tissues, thegastrointestinal system (e.g., mouth, esophagus, stomach, smallintestines, large intestines, rectum), vascular system (e.g., heart,blood vessels and arteries), liver, gallbladder, lymphatic/immune system(e.g., lymph nodes, lymphoid follicles, spleen, thymus, bone marrow),uro-genital system (e.g., kidneys, ureter, bladder, urethra, cervix,Fallopian tubes, ovaries, uterus, vulva, prostate, bulbourethral glands,epidiymis, prostate, seminal vesicles, testicles), musculoskeletalsystem (e.g., skeletal muscles, smooth muscles, bone, cartilage,tendons, ligaments), adipose tissue, mammaries, and the endocrine system(e.g., hypothalamus, pituitary, thyroid, pancreas, adrenal glands).Hence, based on the association of an AspRS polynucleotide orpolypeptide sequence as described herein, these methods may be used toidentify or characterize the tissue or organ from which a cell or otherbiological sample is derived.

Embodiments of the present invention include methods of detecting thepresence or levels of an AspRS polynucleotide or polypeptide referencesequence or a portion thereof to distinguish between or characterize thedevelopmental or differentiation state of the cell. Also included aremethods of differentiating between germ cells, stem cells, and somaticcells. Examples of developmental states include neonatal and adult.Examples of cellular differentiation states include all of the discreetand identifiable stages between a totipotent cell, a pluripotent cell, amultipotent progenitor stem cell and a mature, fully differentiatedcell.

A totipotent cell has total potential, typically arises during sexualand asexual reproduction, and includes and spores and zygotes, though incertain instances cells can dedifferentiate and regain totipotency. Apluripotent cell includes a stem cell that has the potential todifferentiate into any of the three germ layers, including the endoderm(interior stomach lining, gastrointestinal tract, the lungs), themesoderm (muscle, bone, blood, urogenital), and the ectoderm (epidermaltissues and nervous system). Multipotent progenitor cells are typicallycapable of differentiating into a limited number of tissue types.Examples of multipotent cells include, without limitation, hematopoieticstem cells (adult stem cells) from the bone marrow that give rise toimmune cells such as red blood cells, white blood cells, and platelets,mesenchymal stem cells (adult stem cells) from the bone marrow that giverise to stromal cells, fat cells, and various types of bone cells,epithelial stem cells (progenitor cells) that give rise to the varioustypes of skin cells, and muscle satellite cells (progenitor cells) thatcontribute to differentiated muscle tissue. Accordingly, the presence orlevels of particular AspRS polynucleotide or polypeptide sequence can beused to distinguish between or characterize the above-noted cellulardifferentiation states, as compared to a control or a predeterminedlevel.

Embodiments of the present invention include methods of detecting thepresence or levels of an AspRS polynucleotide or polypeptide referencesequence to characterize or diagnose the condition or a cell, tissue,organ, or subject, in which that condition may be characterized ashealthy, diseased, at risk for being diseased, or treated. For suchdiagnostic purposes, the term “diagnostic” or “diagnosed” includesidentifying the presence or nature of a pathologic condition,characterizing the risk of developing such a condition, and/or measuringthe change (or no change) of a pathologic condition in response totherapy. Diagnostic methods may differ in their sensitivity andspecificity. In certain embodiments, the “sensitivity” of a diagnosticassay refers to the percentage of diseased cells, tissues or subjectswhich test positive (percent of “true positives”). Diseased cells,tissues or subjects not detected by the assay are typically referred toas “false negatives.” Cells, tissues or subjects that are not diseasedand which test negative in the assay may be termed “true negatives.” Incertain embodiments, the “specificity” of a diagnostic assay may bedefined as one (1) minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those samples or subjectswithout the disease and which test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis.

In certain instances, the presence or risk of developing a pathologiccondition can be diagnosed by comparing the presence or levels of one ormore selected AspRS polynucleotide or polypeptide reference sequences orportions thereof that correlate with the condition, whether by increasedor decreased levels, as compared to a suitable control. A “suitablecontrol” or “appropriate control” includes a value, level, feature,characteristic, or property determined in a cell or other biologicalsample of a tissue or organism, e.g., a control or normal cell, tissueor organism, exhibiting, for example, normal traits, such as the absenceof the condition. In certain embodiments, a “suitable control” or“appropriate control” is a predefined value, level, feature,characteristic, or property. Other suitable controls will be apparent topersons skilled in the art. Examples of diseases and conditions aredescribed elsewhere herein.

Embodiments of the present invention include AspRS polynucleotide ornucleic acid-based detection techniques, which offer certain advantagesdue to sensitivity of detection. Hence, certain embodiments relate tothe use or detection of AspRS polynucleotides as part of a diagnosticmethod or assay. The presence and/or levels of AspRS polynucleotides maybe measured by any method known in the art, including hybridizationassays such as Northern blot, quantitative or qualitative polymerasechain reaction (PCR), quantitative or qualitative reverse transcriptasePCR (RT-PCR), microarray, dot or slot blots, or in situ hybridizationsuch as fluorescent in situ hybridization (FISH), among others. Certainof these methods are described in greater detail below.

AspRS polynucleotides such as DNA and RNA can be collected and/orgenerated from blood, biological fluids, tissues, organs, cell lines, orother relevant sample using techniques known in the art, such as thosedescribed in Kingston. (2002 Current Protocols in Molecular Biology,Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y. (see, e.g.,as described by Nelson et al. Proc Natl Acad Sci USA, 99: 11890-11895,2002) and elsewhere.

Complementary DNA (cDNA) libraries can be generated using techniquesknown in the art, such as those described in Ausubel et al. (2001Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & JohnWiley & Sons, Inc., NY, N.Y.); Sambrook et al. (1989 Molecular Cloning,Second Ed., Cold Spring Harbor Laboratory, Plainview, N.Y.); Maniatis etal. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview,N.Y.) and elsewhere.

Certain embodiments may employ hybridization methods for detecting AspRSpolynucleotide sequences. Methods for conducting polynucleotidehybridization assays have been well developed in the art. Hybridizationassay procedures and conditions will vary depending on the applicationand are selected in accordance with the general binding methods knownincluding those referred to in: Maniatis et al. Molecular Cloning: ALaboratory Manual (2nd Ed. Cold Spring Harbor, N.Y., 1989); Berger andKimmel Methods in Enzymology, Vol. 152, Guide to Molecular CloningTechniques (Academic Press, Inc., San Diego, Calif., 1987); Young andDavism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying outrepeated and controlled hybridization reactions have been described inU.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623each of which are incorporated herein by reference

Certain embodiments may employ nucleic acid amplification methods fordetecting AspRS polynucleotide sequences. The term “amplification” or“nucleic acid amplification” refers to the production of multiple copiesof a target nucleic acid that contains at least a portion of theintended specific target nucleic acid sequence. The multiple copies maybe referred to as amplicons or amplification products. In certainembodiments, the amplified target contains less than the complete targetgene sequence (introns and exons) or an expressed target gene sequence(spliced transcript of exons and flanking untranslated sequences). Forexample, specific amplicons may be produced by amplifying a portion ofthe target polynucleotide by using amplification primers that hybridizeto, and initiate polymerization from, internal positions of the targetpolynucleotide. Preferably, the amplified portion contains a detectabletarget sequence that may be detected using any of a variety ofwell-known methods.

“Selective amplification” or “specific amplification,” as used herein,refers to the amplification of a target nucleic acid sequence accordingto the present invention wherein detectable amplification of the targetsequence is substantially limited to amplification of target sequencecontributed by a nucleic acid sample of interest that is being testedand is not contributed by target nucleic acid sequence contributed bysome other sample source, e.g., contamination present in reagents usedduring amplification reactions or in the environment in whichamplification reactions are performed.

By “amplification conditions” is meant conditions permitting nucleicacid amplification according to the present invention. Amplificationconditions may, in some embodiments, be less stringent than “stringenthybridization conditions” as described herein. Oligonucleotides used inthe amplification reactions of the present invention hybridize to theirintended targets under amplification conditions, but may or may nothybridize under stringent hybridization conditions. On the other hand,detection probes of the present invention typically hybridize understringent hybridization conditions. Acceptable conditions to carry outnucleic acid amplifications according to the present invention can beeasily ascertained by someone having ordinary skill in the art dependingon the particular method of amplification employed.

Many well-known methods of nucleic acid amplification requirethermocycling to alternately denature double-stranded nucleic acids andhybridize primers; however, other well-known methods of nucleic acidamplification are isothermal. The polymerase chain reaction (U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188), commonly referred toas PCR, uses multiple cycles of denaturation, annealing of primer pairsto opposite strands, and primer extension to exponentially increase copynumbers of the target sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.

As noted above, the term “PCR” refers to multiple amplification cyclesthat selectively amplify a target nucleic acid species. Included arequantitative PCR (qPCR), real-time PCR), reverse transcription PCR(RT-PCR) and quantitative reverse transcription PCR (qRT-PCR) is welldescribed in the art. The term “pPCR” refers to quantitative polymerasechain reaction, and the term “qRT-PCR” refers to quantitative reversetranscription polymerase chain reaction. qPCR and qRT-PCR may be used toamplify and simultaneously quantify a targeted cDNA molecule. It enablesboth detection and quantification of a specific sequence in a cDNA pool,such as a selected AspRS gene or transcript.

The term “real-time PCR” may use DNA-binding dye to bind to alldouble-stranded (ds) DNA in PCR, causing fluorescence of the dye. Anincrease in DNA product during PCR therefore leads to an increase influorescence intensity and is measured at each cycle, thus allowing DNAconcentrations to be quantified. However, dsDNA dyes such as SYBR Greenwill bind to all dsDNA PCR products. Fluorescence is detected andmeasured in the real-time PCR thermocycler, and its geometric increasecorresponding to exponential increase of the product is used todetermine the threshold cycle (“Ct”) in each reaction.

The term “Ct Score” refers to the threshold cycle number, which is thecycle at which PCR amplification has surpassed a threshold level. Ifthere is a higher quantity of mRNA for a particular gene in a sample, itwill cross the threshold earlier than a lowly expressed gene since thereis more starting RNA to amplify. Therefore, a low Ct score indicateshigh gene expression in a sample and a high Ct score is indicative oflow gene expression.

Certain embodiments may employ the ligase chain reaction (Weiss, R.1991, Science 254: 1292), commonly referred to as LCR, which uses twosets of complementary DNA oligonucleotides that hybridize to adjacentregions of the target nucleic acid. The DNA oligonucleotides arecovalently linked by a DNA ligase in repeated cycles of thermaldenaturation, hybridization and ligation to produce a detectabledouble-stranded ligated oligonucleotide product.

In certain embodiments, other techniques may be used to evaluate RNAtranscripts of the transcripts from a particular cDNA library, includingmicroarray analysis (Han, M., et al., Nat Biotechnol, 19: 631-635, 2001;Bao, P., et al., Anal Chem, 74: 1792-1797, 2002; Schena et al., Proc.Natl. Acad. Sci. USA 93:10614-19, 1996; and Heller et al., Proc. Natl.Acad. Sci. USA 94:2150-55, 1997) and SAGE (serial analysis of geneexpression). Like MPSS, SAGE is digital and can generate a large numberof signature sequences. (see e.g., Velculescu, V. E., et al., TrendsGenet, 16: 423-425, 2000; Tuteja R. and Tuteja N. Bioassays. 2004August; 26(8):916-22), although orders of magnitude fewer than that areavailable from techniques such as MPSS.

In certain embodiments, the term “microarray” includes a “nucleic acidmicroarray” having a substrate-bound plurality of nucleic acids,hybridization to each of the plurality of bound nucleic acids beingseparately detectable. The substrate can be solid or porous, planar ornon-planar, unitary or distributed. Nucleic acid microarrays include allthe devices so called in Schena (ed.), DNA Microarrays: A PracticalApproach (Practical Approach Series), Oxford University Press (1999);Nature Genet. 21(1) (suppl.): 1-60 (1999); Schena (ed.), MicroarrayBiochip: Tools and Technology, Eaton Publishing Company/BioTechniquesBooks Division (2000). Nucleic acid microarrays may include asubstrate-bound plurality of nucleic acids in which the plurality ofnucleic acids are disposed on a plurality of beads, rather than on aunitary planar substrate, as described, for example, in Brenner et al.,Proc. Natl. Acad. Sci. USA 97(4): 1665-1670 (2000). Examples of nucleicacid microarrays may be found in U.S. Pat. Nos. 6,391,623, 6,383,754,6,383,749, 6,380,377, 6,379,897, 6,376,191, 6,372,431, 6,351,7126,344,316, 6,316,193, 6,312,906, 6,309,828, 6,309,824, 6,306,643,6,300,063, 6,287,850, 6,284,497, 6,284,465, 6,280,954, 6,262,216,6,251,601, 6,245,518, 6,263,287, 6,251,601, 6,238,866, 6,228,575,6,214,587, 6,203,989, 6,171,797, 6,103,474, 6,083,726, 6,054,274,6,040,138, 6,083,726, 6,004,755, 6,001,309, 5,958,342, 5,952,180,5,936,731, 5,843,655, 5,814,454, 5,837,196, 5,436,327, 5,412,087, and5,405,783, the disclosures of which are incorporated by reference.

Additional examples include nucleic acid arrays that are commerciallyavailable from Affymetrix (Santa Clara, Calif.) under the brand nameGeneChip™. Further exemplary methods of manufacturing and using arraysare provided in, for example, U.S. Pat. Nos. 7,028,629; 7,011,949;7,011,945; 6,936,419; 6,927,032; 6,924,103; 6,921,642; and 6,818,394.

The present invention as related to arrays and microarrays alsocontemplates many uses for polymers attached to solid substrates. Theseuses include gene expression monitoring, profiling, library screening,genotyping and diagnostics. Gene expression monitoring and profilingmethods and methods useful for gene expression monitoring and profilingare shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860,6,040,138, 6,177,248 and 6,309,822. Genotyping and uses therefore areshown in U.S. Ser. Nos. 10/442,021, 10/013,598 (U.S. Application No.2003/0036069), and U.S. Pat. Nos. 5,925,525, 6,268,141, 5,856,092,6,267,152, 6,300,063, 6,525,185, 6,632,611, 5,858,659, 6,284,460,6,361,947, 6,368,799, 6,673,579 and 6,333,179. Other methods of nucleicacid amplification, labeling and analysis that may be used incombination with the methods disclosed herein are embodied in U.S. Pat.Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.

As will be apparent to persons skilled in the art, certain embodimentsmay employ oligonucleotides, such as primers or probes, foramplification or detection, as described herein. While the design andsequence of oligonucleotides depends on their function as describedherein, several variables are generally taken into account. Among themost relevant are: length, melting temperature (Tm), specificity,complementarity with other oligonucleotides in the system, G/C content,polypyrimidine (T, C) or polypurine (A, G) stretches, and the 3′-endsequence.

Certain embodiments therefore include methods for detecting a targetAspRS polynucleotide in a sample, typically wherein the polynucleotidecomprises the sequence of a reference AspRS polynucleotide describedherein, comprising a) hybridizing the sample with a probe comprising asequence complementary to the target polynucleotide in the sample, andwhich probe specifically hybridizes to said target polynucleotide, underconditions whereby a hybridization complex is formed between said probeand said target polynucleotide or fragments thereof, and b) detectingthe presence or absence of said hybridization complex, and optionally,if present, the amount thereof. Also included are methods for detectinga target AspRS polynucleotide in a sample, the polynucleotide comprisingthe sequence of a reference AspRS polynucleotide, as described herein,comprising a) amplifying the target polynucleotide or fragment thereof,and b) detecting the presence or absence of said amplified targetpolynucleotide or fragment thereof, and, optionally, if present, theamount thereof.

Embodiments of the present invention include a variety of AspRSpolypeptide-based detection techniques, including antibody-baseddetection techniques. Included in these embodiments are the use of AspRSpolypeptides to generate antibodies or other binders, which may then beused in diagnostic methods and compositions to detect or quantitateselected AspRS polypeptides in a cell or other biological sample,typically from a subject.

Certain embodiments may employ standard methodologies such as westernblotting and immunoprecipitation, enzyme-linked immunosorbent assays(ELISA), flow cytometry, and immunofluorescence assays (IFA). Thesewell-known methods typically utilize one or more monoclonal orpolyclonal antibodies as described herein that specifically bind to aselected AspRS polypeptide of the invention, or a unique region of thatAspRS polypeptide, and generally do not bind significantly to otherAspRS polypeptides, such as a full-length AspRS polypeptide. In certainembodiments, the unique region of the AspRS polypeptide may be encodedby a unique splice junction or a particular three-dimensional structureof a newly identified alternate splice variant or protein fragment, suchas a proteolytic fragment.

Certain embodiments may employ “arrays,” such as “microarrays.” Incertain embodiments, a “microarray” may also refer to a “peptidemicroarray” or “protein microarray” having a substrate-bound collectionor plurality of polypeptides, the binding to each of the plurality ofbound polypeptides being separately detectable. Alternatively, thepeptide microarray may have a plurality of binders, including but notlimited to monoclonal antibodies, polyclonal antibodies, phage displaybinders, yeast 2 hybrid binders, and aptamers, which can specificallydetect the binding of the AspRS polypeptides described herein. The arraymay be based on autoantibody detection of these AspRS polypeptides, asdescribed, for example, in Robinson et al., Nature Medicine 8(3):295-301(2002). Examples of peptide arrays may be found in WO 02/31463, WO02/25288, WO 01/94946, WO 01/88162, WO 01/68671, WO 01/57259, WO00/61806, WO 00/54046, WO 00/47774, WO 99/40434, WO 99/39210, and WO97/42507 and U.S. Pat. Nos. 6,268,210, 5,766,960, and 5,143,854, each ofwhich are incorporated by reference.

Certain embodiments may employ MS or other molecular weight-basedmethods for diagnostically detecting AspRS polypeptide sequences. Massspectrometry (MS) refers generally to an analytical technique fordetermining the elemental composition of a sample or molecule. MS mayalso be used for determining the chemical structures of molecules, suchas peptides and other chemical compounds.

Generally, the MS principle consists of ionizing chemical compounds togenerate charged molecules or molecule fragments, and then measuringtheir mass-to-charge ratios. In an illustrative MS procedure: a sampleis loaded onto the MS instrument, and undergoes vaporization, thecomponents of the sample are ionized by one of a variety of methods(e.g., by impacting them with an electron beam), which results in theformation of positively charged particles, the positive ions are thenaccelerated by a magnetic field, computations are performed on themass-to-charge ratio (m/z) of the particles based on the details ofmotion of the ions as they transit through electromagnetic fields, and,detection of the ions, which in step prior were sorted according to m/z.

An illustrative MS instruments has three modules: an ion source, whichconverts gas phase sample molecules into ions (or, in the case ofelectrospray ionization, move ions that exist in solution into the gasphase); a mass analyzer, which sorts the ions by their masses byapplying electromagnetic fields; and a detector, which measures thevalue of an indicator quantity and thus provides data for calculatingthe abundances of each ion present.

The MS technique has both qualitative and quantitative uses, includingidentifying unknown compounds, determining the isotopic composition ofelements in a molecule, and determining the structure of a compound byobserving its fragmentation. Other uses include quantifying the amountof a compound in a sample or studying the fundamentals of gas phase ionchemistry (the chemistry of ions and neutrals in a vacuum). Accordingly,MS techniques may be used according to any of the methods providedherein to measure the presence or levels of an AspRS polypeptide of theinvention in a biological sample, and to compare those levels to acontrol sample or a pre-determined value.

B. Discovery of Compounds and Therapeutic Agents

Certain embodiments relate to the use of AspRS polypeptide or AspRSpolynucleotide references sequences in drug discovery, typically toidentify agents that modulate one or more of the non-canonicalactivities of the reference AspRS. For example, certain embodimentsinclude methods of identifying one or more “binding partners” of anAspRS reference polypeptide, or a polypeptide that comprises an AspRSreference sequence such as a cellular protein or other host moleculethat associates with the AspRS polypeptide and participates in itsnon-canonical activity or activities. Also included are methods ofidentifying a compound (e.g., polypeptide) or other agent that agonizesor antagonizes the non-canonical activity of an AspRS referencepolypeptide or active variant thereof, such as by interacting with theAspRS polypeptide and/or one or more of its cellular binding partners.

Certain embodiments therefore include methods of identifying a bindingpartner of an AspRS reference polypeptide, comprising a) combining theAspRS polypeptide with a biological sample under suitable conditions,and b) detecting specific binding of the AspRS polypeptide to a bindingpartner, thereby identifying a binding partner that specifically bindsto the AspRS reference polypeptide. Also included are methods ofscreening for a compound that specifically binds to an AspRS referencepolypeptide or a binding partner of the AspRS polypeptide, comprising a)combining the polypeptide or the binding partner with at least one testcompound under suitable conditions, and b) detecting binding of thepolypeptide or the binding partner to the test compound, therebyidentifying a compound that specifically binds to the polypeptide or itsbinding partner. In certain embodiments, the compound is a polypeptideor peptide. In certain embodiments, the compound is a small molecule orother (e.g., non-biological) chemical compound. In certain embodiments,the compound is a peptide mimetic.

Any method suitable for detecting protein-protein interactions may beemployed for identifying cellular proteins that interact with an AspRSreference polypeptide, interact with one or more of its cellular bindingpartners, or both. Examples of traditional methods that may be employedinclude co-immunoprecipitation, cross-linking, and co-purificationthrough gradients or chromatographic columns of cell lysates or proteinsobtained from cell lysates, mainly to identify proteins in the lysatethat interact with the AspRS polypeptide.

In these and related embodiments, at least a portion of the amino acidsequence of a protein that interacts with an AspRS polypeptide or itsbinding partner can be ascertained using techniques well known to thoseof skill in the art, such as via the Edman degradation technique. See,e.g., Creighton Proteins: Structures and Molecular Principles, W. H.Freeman & Co., N.Y., pp. 34 49, 1983. The amino acid sequence obtainedmay be used as a guide for the generation of oligonucleotide mixturesthat can be used to screen for gene sequences encoding such proteins.Screening may be accomplished, for example, by standard hybridization orPCR techniques, as described herein and known in the art. Techniques forthe generation of oligonucleotide mixtures and the screening are wellknown. See, e.g., Ausubel et al. Current Protocols in Molecular BiologyGreen Publishing Associates and Wiley Interscience, N.Y., 1989; andInnis et al., eds. PCR Protocols: A Guide to Methods and ApplicationsAcademic Press, Inc., New York, 1990.

Additionally, methods may be employed in the simultaneous identificationof genes that encode the binding partner or other polypeptide. Thesemethods include, for example, probing expression libraries, in a mannersimilar to the well known technique of antibody probing of lambda-gt11libraries, using labeled AspRS protein, or another polypeptide, peptideor fusion protein, e.g., a variant AspRS polypeptide or AspRS domainfused to a marker (e.g., an enzyme, fluor, luminescent protein, or dye),or an Ig-Fc domain.

One method that detects protein interactions in vivo is the two-hybridsystem. An example of this system has been described (Chien et al., PNASUSA 88:9578 9582, 1991) and is commercially available from Clontech(Palo Alto, Calif.). In certain instances, the two-hybrid system orother such methodology may be used to screen activation domain librariesfor proteins that interact with the “bait” gene product. By way ofexample, and not by way of limitation, an AspRS reference polypeptide orvariant may be used as the bait gene product. An AspRS binding partnermay also be used as a “bait” gene product. Total genomic or cDNAsequences are fused to the DNA encoding an activation domain. Thislibrary and a plasmid encoding a hybrid of a bait AspRS gene productfused to the DNA-binding domain are co-transformed into a yeast reporterstrain, and the resulting transformants are screened for those thatexpress the reporter gene.

Also included are three-hybrid systems, which allow the detection ofRNA-protein interactions in yeast. See, e.g., Hook et al., RNA.11:227-233, 2005. Accordingly, these and related methods can be used toidentify a cellular binding partner of an AspRS polypeptide. These andrelated methods can also be used to identify other compounds such asbinding agents or nucleic acids that interact with the AspRSpolypeptide, its cellular binding partner, or both.

As noted above, once isolated, binding partners can be identified andcan, in turn, be used in conjunction with standard techniques toidentify proteins or other compounds with which it interacts. Certainembodiments thus relate to methods of screening for a compound thatspecifically binds to the binding partner of an AspRS referencepolypeptide, comprising a) combining the binding partner with at leastone test compound under suitable conditions, and b) detecting binding ofthe binding partner to the test compound, thereby identifying a compoundthat specifically binds to the binding partner. In certain embodiments,the test compound is a polypeptide. In certain embodiments, the testcompound is a chemical compound, such as a small molecule compound orpeptide mimetic.

Certain embodiments include methods of screening for a compound thatmodulates the activity of an AspRS reference polypeptide, comprising a)combining the polypeptide with at least one test compound underconditions permissive for the activity of the polypeptide, b) assessingthe activity of the polypeptide in the presence of the test compound,and c) comparing the activity of the polypeptide in the presence of thetest compound with the activity of the polypeptide in the absence of thetest compound, wherein a change in the activity of the polypeptide inthe presence of the test compound is indicative of a compound thatmodulates the activity of the polypeptide.

Certain embodiments include methods of screening for a compound thatmodulates the activity of a binding partner of an AspRS referencepolypeptide, comprising a) combining the polypeptide with at least onetest compound under conditions permissive for the activity of thebinding partner, b) assessing the activity of the binding partner in thepresence of the test compound, and c) comparing the activity of thebinding partner in the presence of the test compound with the activityof the binding partner in the absence of the test compound, wherein achange in the activity of the binding partner in the presence of thetest compound is indicative of a compound that modulates the activity ofthe binding partner. Typically, these and related embodiments includeassessing a selected non-canonical activity that is associated with theAspRS polypeptide or its binding partner. Included are in vitro and invivo conditions, such as cell culture conditions.

Certain embodiments include methods of screening a compound foreffectiveness as a full or partial agonist of an AspRS referencepolypeptide or an active fragment or variant thereof, comprising a)exposing a sample comprising the polypeptide to a compound, and b)detecting agonist activity in the sample, typically by measuring anincrease in the non-canonical activity of the AspRS polypeptide. Certainmethods include a) exposing a sample comprising a binding partner of theAspRS polypeptide to a compound, and b) detecting agonist activity inthe sample, typically by measuring an increase in the selectednon-canonical activity of the AspRS polypeptide. Certain embodimentsinclude compositions that comprise an agonist compound identified by themethod and a pharmaceutically acceptable carrier or excipient.

Also included are methods of screening a compound for effectiveness as afull or partial antagonist of an AspRS reference polypeptide, comprisinga) exposing a sample comprising the polypeptide to a compound, and b)detecting antagonist activity in the sample, typically by measuring adecrease in the non-canonical activity of the AspRS polypeptide. Certainmethods include a) exposing a sample comprising a binding partner of theAspRS polypeptide to a compound, and b) detecting antagonist activity inthe sample, typically by measuring a decrease in the selectednon-canonical activity of the AspRS polypeptide. Certain embodimentsinclude compositions that comprise an antagonist compound identified bythe method and a pharmaceutically acceptable carrier or excipient.

In certain embodiments, in vitro systems may be designed to identifycompounds capable of interacting with or modulating an AspRS referencesequence or its binding partner. Certain of the compounds identified bysuch systems may be useful, for example, in modulating the activity ofthe pathway, and in elaborating components of the pathway itself. Theymay also be used in screens for identifying compounds that disruptinteractions between components of the pathway; or may disrupt suchinteractions directly. One exemplary approach involves preparing areaction mixture of the AspRS polypeptide and a test compound underconditions and for a time sufficient to allow the two to interact andbind, thus forming a complex that can be removed from and/or detected inthe reaction mixture

In vitro screening assays can be conducted in a variety of ways. Forexample, an AspRS polypeptide, a cellular binding partner, or testcompound(s) can be anchored onto a solid phase. In these and relatedembodiments, the resulting complexes may be captured and detected on thesolid phase at the end of the reaction. In one example of such a method,the AspRS polypeptide and/or its binding partner are anchored onto asolid surface, and the test compound(s), which are not anchored, may belabeled, either directly or indirectly, so that their capture by thecomponent on the solid surface can be detected. In other examples, thetest compound(s) are anchored to the solid surface, and the AspRSpolypeptide and/or its binding partner, which are not anchored, arelabeled or in some way detectable. In certain embodiments, microtiterplates may conveniently be utilized as the solid phase. The anchoredcomponent (or test compound) may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

To conduct an exemplary assay, the non-immobilized component istypically added to the coated surface containing the anchored component.After the reaction is complete, un-reacted components are removed (e.g.,by washing) under conditions such that any specific complexes formedwill remain immobilized on the solid surface. The detection of complexesanchored on the solid surface can be accomplished in a number of ways.For instance, where the previously non-immobilized component ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the previously non-immobilizedcomponent is not pre-labeled, an indirect label can be used to detectcomplexes anchored on the surface; e.g., using a labeled antibodyspecific for the previously non-immobilized component (the antibody, inturn, may be directly labeled or indirectly labeled with a labeledanti-Ig antibody).

Alternatively, the presence or absence of binding of a test compound canbe determined, for example, using surface plasmon resonance (SPR) andthe change in the resonance angle as an index, wherein an AspRSpolypeptide or a cellular binding partner is immobilized onto thesurface of a commercially available sensorchip (e.g., manufactured byBiacore™) according to a conventional method, the test compound iscontacted therewith, and the sensorchip is illuminated with a light of aparticular wavelength from a particular angle. The binding of a testcompound can also be measured by detecting the appearance of a peakcorresponding to the test compound by a method wherein an AspRSpolypeptide or a cellular binding partner is immobilized onto thesurface of a protein chip adaptable to a mass spectrometer, a testcompound is contacted therewith, and an ionization method such asMALDI-MS, ESI-MS, FAB-MS and the like is combined with a massspectrometer (e.g., double-focusing mass spectrometer, quadrupole massspectrometer, time-of-flight mass spectrometer, Fourier transformationmass spectrometer, ion cyclotron mass spectrometer and the like).

In certain embodiments, cell-based assays, membrane vesicle-basedassays, or membrane fraction-based assays can be used to identifycompounds that modulate interactions in the non-canonical pathway of theselected AspRS polypeptide. To this end, cell lines that express anAspRS polypeptide and/or a binding partner, or a fusion proteincontaining a domain or fragment of such proteins (or a combinationthereof), or cell lines (e.g., COS cells, CHO cells, HEK293 cells, Helacells etc.) that have been genetically engineered to express suchprotein(s) or fusion protein(s) can be used. Test compound(s) thatinfluence the non-canonical activity can be identified by monitoring achange (e.g., a statistically significant change) in that activity ascompared to a control or a predetermined amount.

For embodiments that relate to antisense and RNAi agents, for example,also included are methods of screening a compound for effectiveness inaltering expression of an AspRS reference polynucleotide, comprising a)exposing a sample comprising the AspRS reference polynucleotide to acompound such as a potential antisense oligonucleotide, and b) detectingaltered expression of the AspRS polynucleotide. In certain non-limitingexamples, these and related embodiments can be employed in cell-basedassays or in cell-free translation assays, according to routinetechniques in the art. Also included are the antisense and RNAi agentsidentified by such methods.

Also included are any of the above methods, or other screening methodsknown in the art, which are adapted for high-throughput screening (HTS).HTS typically uses automation to run a screen of an assay against alibrary of candidate compounds, for instance, an assay that measures anincrease or a decrease in a non-canonical activity, as described herein.

C. Methods of Treatment

In another aspect, the present invention relates to methods of using thecompositions of the present invention for treating a cell, tissue orsubject with a composition as described herein. The cells or tissue thatmay be modulated by the present invention are preferably mammaliancells, or more preferably human cells. Such cells can be of a healthystate or of a diseased state.

Accordingly, the AspRS agents described herein, including AspRSpolypeptides, AspRS polynucleotides, AspRS polynucleotide-based vectors,antisense oligonucleotides, RNAi agents, as well as binding agents suchas peptides, antibodies and antigen-binding fragments, peptide mimeticsand other small molecules, can be used to treat a variety ofnon-limiting diseases or conditions associated with the non-canonicalactivities of a reference AspRS. Examples of such non-canonicalactivities include modulation of cell proliferation, modulation of cellmigration, modulation of cell differentiation (e.g., hematopoiesis),modulation of apoptosis or other forms of cell death, modulation of cellsignaling, modulation of angiogenesis, modulation of cell binding,modulation of cellular metabolism, modulation of cytokine production oractivity, modulation of cytokine receptor activity, modulation ofinflammation, and the like.

Included are polynucleotide-based therapies, such as antisense therapiesand RNAi interference therapies, which typically relate to reducing theexpression of a target molecule, such as a particular splice variant ofan AspRS polypeptide or a cellular binding partner of an AspRSpolypeptide, which otherwise contributes to its non-canonical activity.Antisense or RNAi therapies typically antagonize the non-canonicalactivity, such as by reducing expression of the AspRS referencepolypeptide. Also included are polypeptides, antibodies, peptidemimetics, or other small molecule-based therapies, which either agonizeor antagonize the non-canonical activity of an AspRS referencepolypeptide, such as by interacting directly with the AspRS polypeptide,its cellular binding partner(s), or both.

In certain embodiments, for example, methods are provided for modulatingtherapeutically relevant cellular activities including, but not limitedto, cellular metabolism, cell differentiation, cell proliferation, celldeath, cell mobilization, cell migration, gene transcription, mRNAtranslation, cell impedance, cytokine production, and the like,comprising contacting a cell with an AspRS composition as describedherein. Accordingly, the AspRS compositions may be employed in treatingessentially any cell or tissue or subject that would benefit frommodulation of one or more such activities.

The AspRS compositions may also be used in any of a number oftherapeutic contexts including, for example, those relating to thetreatment or prevention of neoplastic diseases, immune system diseases(e.g., autoimmune diseases and inflammation), infectious diseases,metabolic diseases, neuronal/neurological diseases,muscular/cardiovascular diseases, diseases associated with aberranthematopoiesis, diseases associated with aberrant angiogenesis, diseasesassociated with aberrant cell survival, and others.

For example, in certain illustrative embodiments, the AspRS compositionsof the invention may be used to modulate angiogenesis, e.g., viamodulation of endothelial cell proliferation and/or signaling.Endothelial cell proliferation and/or cell signaling may be monitoredusing an appropriate cell line (e.g., Human microvascular endotheliallung cells (HMVEC-L) and Human umbilical vein endothelial cells(HUVEC)), and using an appropriate assay (e.g., endothelial cellmigration assays, endothelial cell proliferation assays, tube-formingassays, matrigel plug assays, etc.), many of which are known andavailable in the art.

Therefore, in related embodiments, the compositions of the invention maybe employed in the treatment of essentially any cell or tissue orsubject that would benefit from modulation of angiogenesis. For example,in some embodiments, a cell or tissue or subject experiencing orsusceptible to angiogenesis (e.g., an angiogenic condition) may becontacted with a suitable composition of the invention to inhibit anangiogenic condition. In other embodiments, a cell or tissueexperiencing or susceptible to insufficient angiogenesis (e.g., anangiostatic condition) may be contacted with an appropriate compositionof the invention in order to interfere with angiostatic activity and/orpromote angiogenesis.

Illustrative examples of angiogenic conditions include, but are notlimited to, age-related macular degeneration (AMD), cancer (both solidand hematologic), developmental abnormalities (organogenesis), diabeticblindness, endometriosis, ocular neovascularization, psoriasis,rheumatoid arthritis (RA), and skin disclolorations (e.g., hemangioma,nevus flammeus or nevus simplex). Examples of anti-angiogenic conditionsinclude, but are not limited to, cardiovascular disease, restenosis,tissue damage after reperfusion of ischemic tissue or cardiac failure,chronic inflammation and wound healing.

The compositions of the invention may also be useful as immunomodulatorsfor treating anti- or pro-inflammatory indications by modulating thecells that mediate, either directly or indirectly, autoimmune and/orinflammatory disease, conditions and disorders. The utility of thecompositions of the invention as immunomodulators can be monitored usingany of a number of known and available techniques in the art including,for example, migration assays (e.g., using leukocytes or lymphocytes),cytokine production assays, or cell viability assays (e.g., usingB-cells, T-cells, monocytes or NK cells).

“Inflammation” refers generally to the biological response of tissues toharmful stimuli, such as pathogens, damaged cells (e.g., wounds), andirritants. The term “inflammatory response” refers to the specificmechanisms by which inflammation is achieved and regulated, including,merely by way of illustration, immune cell activation or migration,cytokine production, vasodilation, including kinin release,fibrinolysis, and coagulation, among others described herein and knownin the art. Ideally, inflammation is a protective attempt by the body toboth remove the injurious stimuli and initiate the healing process forthe affected tissue or tissues. In the absence of inflammation, woundsand infections would never heal, creating a situation in whichprogressive destruction of the tissue would threaten survival. On theother hand, excessive or chronic inflammation may associate with avariety of diseases, such as hay fever, atherosclerosis, and rheumatoidarthritis, among others described herein and known in the art.

Clinical signs of chronic inflammation are dependent upon duration ofthe illness, inflammatory lesions, cause and anatomical area affected.(see, e.g., Kumar et al., Robbins Basic Pathology-8^(th) Ed., 2009Elsevier, London; Miller, L M, Pathology Lecture Notes, AtlanticVeterinary College, Charlottetown, PEI, Canada). Chronic inflammation isassociated with a variety of pathological conditions or diseases,including, for example, allergies, Alzheimer's disease, anemia, aorticvalve stenosis, arthritis such as rheumatoid arthritis andosteoarthritis, cancer, congestive heart failure, fibromyalgia,fibrosis, heart attack, kidney failure, lupus, pancreatitis, stroke,surgical complications, inflammatory lung disease, inflammatory boweldisease, atherosclerosis, neurological disorders, diabetes, metabolicdisorders, obesity, and psoriasis, among others described herein andknown in the art. Hence, AspRS compositions may be used to treat ormanage chronic inflammation, modulate any of one or more of theindividual chronic inflammatory responses, or treat any one or morediseases or conditions associated with chronic inflammation.

Certain specific inflammatory responses include cytokine production andactivity, and related pathways. For instance, certain exemplaryembodiments relate to modulating cell-signaling through nuclearfactor-kB (NF-kB), such as by increasing the downstream activities ofthis transcription factor. In certain instances, increases in NF-kBactivity can lead to increases in cytokine signaling or activity, suchas pro-inflammatory cytokines (e.g., TNF-α), and anti-inflammatorycytokines (e.g., IL-10).

Criteria for assessing the signs and symptoms of inflammatory and otherconditions, including for purposes of making differential diagnosis andalso for monitoring treatments such as determining whether atherapeutically effective dose has been administered in the course oftreatment, e.g., by determining improvement according to acceptedclinical criteria, will be apparent to those skilled in the art and areexemplified by the teachings of e.g., Berkow et al., eds., The MerckManual, 16^(th) edition, Merck and Co., Rahway, N.J., 1992; Goodman etal., eds., Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 10^(th) edition, Pergamon Press, Inc., Elmsford, N.Y.,(2001); Avery's Drug Treatment: Principles and Practice of ClinicalPharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd., Williamsand Wilkins, Baltimore, Md. (1987); Ebadi, Pharmacology, Little, Brownand Co., Boston, (1985); Osolci al., eds., Remington's PharmaceuticalSciences, 18^(th) edition, Mack Publishing Co., Easton, Pa. (1990);Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk,Conn. (1992).

Also included are methods of modulating an immune response, such as aninnate immune response. As used herein, the term “immune response”includes a measurable or observable reaction to an antigen, vaccinecomposition, or immunomodulatory molecule mediated by one or more cellsof the immune system. An immune response typically begins with anantigen or immunomodulatory molecule binding to an immune system cell. Areaction to an antigen or immunomodulatory molecule may be mediated bymany cell types, including a cell that initially binds to an antigen orimmunomodulatory molecule and cells that participate in mediating aninnate, humoral, cell-mediated immune response.

An “innate immune response,” as used herein, may involve binding ofpathogen-associated molecular patterns (PAMPs) or an AspRS polypeptideto cell surface receptors, such as toll-like receptors. Activation oftoll-like receptors and Ipaf-signaling pathways in response to PAMPs orother signals leads to the production of immunomodulatory molecules,such as cytokines and co-stimulatory molecules, which induce and/orenhance an immune response. Cells involved in the innate immune responseinclude, for example, dendritic cells, macrophages, natural killercells, and neutrophils, among others.

Certain embodiments relate to increasing an innate immune response.Other embodiments relate to decreasing an innate immune response. Incertain aspects, an innate immune response is mediated by one or moretoll-like receptors (TLRs), such as TLR2 and/or TLR4. Certain AspRSpolypeptides of the invention bind to TLRS such as TLR2 and/or TLR4.TLRs recognize PAMPs that distinguish infectious agents from self andmediating the production of immunomodulatory molecules, such ascytokines, necessary for the development of effective adaptive immunity(Aderem, A and Ulevitch, R. J. Nature 406: 782-787 (2000) andBrightbill, H. D., Immunology 101: 1-10 (2000), herein incorporated byreference). Members of the toll-like receptor family recognize a varietyof antigen types and can discriminate between pathogens. For example,TLR2 recognizes various fungal, Gram-positive, and mycobacterialcomponents, TLR4 recognizes the Gram-negative product lipopolysaccharide(LPS), and TLR9 recognizes nucleic acids such as CpG repeats inbacterial DNA.

AspRS compositions that stimulate innate immunity (e.g., via TLR2 and/rTLR4) can be useful in the treatment of a wide variety of conditions,either alone or in combination with other therapies. Specific examplesof such conditions include infectious diseases, such as bacterial,viral, and parasitic infectious diseases. AspRS compositions thatstimulate innate immunity can also be useful as vaccine adjuvants, toenhance a subject's immune response to the primary antigen, whether in alive, attenuated, or other type of vaccine.

Examples of viral infectious diseases or agents (and their correspondingvaccines) include, but are not limited to, Hepatitis A, Hepatitis B,Hepatitis C, Hepatitis E, Caliciviruses associated diarrhoea, Rotavirusdiarrhoea, Haemophilus influenzae B pneumonia and invasive disease,influenza, measles, mumps, rubella, Parainfluenza associated pneumonia,Respiratory syncytial virus (RSV) pneumonia, Severe Acute RespiratorySyndrome (SARS), Human papillomavirus, Herpes simplex type 2 genitalulcers, HIV/AIDS, Dengue Fever, Japanese encephalitis, Tick-borneencephalitis, West-Nile virus associated disease, Yellow Fever,Epstein-Barr virus, Lassa fever, Crimean-Congo haemorrhagic fever, Ebolahaemorrhagic fever, Marburg haemorrhagic fever, Rabies, Rift Valleyfever, Smallpox, leprosy, upper and lower respiratory infections,poliomyelitis, among others described elsewhere herein.

Examples of bacterial infections disease or agents include, but are notlimited to, Bacillus antracis, Borellia burgdorferi, Brucella abortus,Brucella canus, Brucella melitensis, Brucella suis, Campylobacterjejuni, Chlamydia pneumoniae, Chlamydia psitacci, Chlamydia trachomatis,Clostridium botulinum, C. difficile, C. perfringens, C. tetani,Corynebacterium diphtheriae (i.e., diphtheria), Enterococcus,Escherichia coli, Haemophilus influenza, Helicobacter pylori, Legionellapneumophila, Leptospira, Listeria monocytogenes, Mycobacterium leprae,M. tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhea, N.meningitidis, Pseudomonas aeruginosa, Rickettsia recketisii, Salmonellatyphi, S. typhimurium, Shigella sonnei, Staphylococcus aureus, S.epidermidis, S. saprophyticus, Streptococcus agalactiae, S. pneumoniae,S. pyogenes, Treponema pallidum, Vibrio cholera, Yersinia pestis,Bordatella pertussis, and otitis media (e.g., often caused byStreptococcus pneumoniae, Haemophilus influenzae, or Moraxellacatarrhalis), among others described elsewhere herein.

Examples of parasitic infectious diseases include, but are not limitedto, Amoebiasis (e.g., Entemoeba histolytica), Hookworm Disease (e.g.,nematode parasites such as Necator americanus and Ancylostomaduodenale), Leishmaniasis, Malaria (four species of the protozoanparasite Plasmodium; P. falciparum, P. vivax, P. ovale, and P.malariae), Schistosomiasis (parasitic Schistosoma; S. mansoni, S.haematobium, and S. japonicum), Onchocerca volvulus (River blindness),Trypanosoma cruzi (Chagas disease/American sleeping sickness), andDracunculus medinensis, lymphatic filariasis.

Certain AspRS compositions may be useful in the treatment or reductionof endotoxic shock, which often results from exposure to foreignantigens, such as lipopolysaccharide (LPS). Because endotoxic shock canbe mediated by TLR signaling, and naturally-occurring endogenous AspRSfragments may stimulate TLRs, certain of the binding agents, antisenseagents, or RNAi agents provided herein may render a subject moreresistant to endotoxic shock by antagonizing or otherwise reducing theendogenous AspRS fragment-mediated stimulation of TLR2 and/or TLR4.

Also included are methods of treating immune diseases. Illustrativeimmune system diseases, disorders or conditions that may be treatedaccording to the present invention include, but are not limited to,primary immunodeficiencies, immune-mediated thrombocytopenia, Kawasakisyndrome, bone marrow transplant (for example, recent bone marrowtransplant in adults or children), chronic B cell lymphocytic leukemia,HIV infection (for example, adult or pediatric HIV infection), chronicinflammatory demyelinating polyneuropathy, post-transfusion purpura, andthe like.

Additionally, further diseases, disorders and conditions includeGuillain-Barre syndrome, anemia (for example, anemia associated withparvovirus B19, patients with stable multiple myeloma who are at highrisk for infection (for example, recurrent infection), autoimmunehemolytic anemia (for example, warm-type autoimmune hemolytic anemia),thrombocytopenia (for example, neonatal thrombocytopenia), andimmune-mediated neutropenia), transplantation (for example,cytomegalovirus (CMV)-negative recipients of CMV-positive organs),hypogammaglobulinemia (for example, hypogammaglobulinemic neonates withrisk factor for infection or morbidity), epilepsy (for example,intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis(for example, decompensation in myasthenia gravis), dermatomyositis, andpolymyositis.

Further autoimmune diseases, disorders and conditions include but arenot limited to, autoimmune hemolytic anemia, autoimmune neonatalthrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (forexample, IgA nephropathy), multiple sclerosis, neuritis, uveitisophthalmia, polyendocrinopathies, purpura (for example,Henloch-Scoenlein purpura), Reiter's disease, stiff-man syndrome,autoimmune pulmonary inflammation, Guillain-Barre Syndrome, insulindependent diabetes mellitus, and autoimmune inflammatory eye disease.

Additional autoimmune diseases, disorders or conditions include, but arenot limited to, autoimmune thyroiditis; hypothyroidism, includingHashimoto's thyroiditis and thyroiditis characterized, for example, bycell-mediated and humoral thyroid cytotoxicity; SLE (which is oftencharacterized, for example, by circulating and locally generated immunecomplexes); Goodpasture's syndrome (which is often characterized, forexample, by anti-basement membrane antibodies); pemphigus (which isoften characterized, for example, by epidermal acantholytic antibodies);receptor autoimmunities such as, for example, Graves' disease (which isoften characterized, for example, by antibodies to a thyroid stimulatinghormone receptor; myasthenia gravis, which is often characterized, forexample, by acetylcholine receptor antibodies); insulin resistance(which is often characterized, for example, by insulin receptorantibodies); autoimmune hemolytic anemia (which is often characterized,for example, by phagocytosis of antibody-sensitized red blood cells);and autoimmune thrombocytopenic purpura (which is often characterized,for example, by phagocytosis of antibody-sensitized platelets).

Further autoimmune diseases, disorders or conditions include, but arenot limited to, rheumatoid arthritis (which is often characterized, forexample, by immune complexes in joints); scleroderma with anti-collagenantibodies (which is often characterized, for example, by nucleolar andother nuclear antibodies); mixed connective tissue disease, (which isoften characterized, for example, by antibodies to extractable nuclearantigens, for example, ribonucleoprotein); polymyositis/dermatomyositis(which is often characterized, for example, by nonhistone anti-nuclearantibodies); pernicious anemia (which is often characterized, forexample, by antiparietal cell, antimicrosome, and anti-intrinsic factorantibodies); idiopathic Addison's disease (which is often characterized,for example, by humoral and cell-mediated adrenal cytotoxicity);infertility (which is often characterized, for example, byantispennatozoal antibodies); glomerulonephritis (which is oftencharacterized, for example, by glomerular basement membrane antibodiesor immune complexes); by primary glomerulonephritis, by IgA nephropathy;bullous pemphigoid (which is often characterized, for example, by IgGand complement in the basement membrane); Sjogren's syndrome (which isoften characterized, for example, by multiple tissue antibodies and/orthe specific nonhistone antinuclear antibody (SS-B)); diabetes mellitus(which is often characterized, for example, by cell-mediated and humoralislet cell antibodies); and adrenergic drug resistance, includingadrenergic drug resistance with asthma or cystic fibrosis (which isoften characterized, for example, by beta-adrenergic receptorantibodies).

Still further autoimmune diseases, disorders or conditions include, butare not limited to chronic active hepatitis (which is oftencharacterized, for example by smooth muscle antibodies); primary biliarycirrhosis (which is often characterized, for example, byanti-mitochondrial antibodies); other endocrine gland failure (which ischaracterized, for example, by specific tissue antibodies in somecases); vitiligo (which is often characterized, for example, byanti-melanocyte antibodies); vasculitis (which is often characterized,for example, by immunoglobulin and complement in vessel walls and/or lowserum complement); post-myocardial infarction conditions (which areoften characterized, for example, by anti-myocardial antibodies);cardiotomy syndrome (which is often characterized, for example, byanti-myocardial antibodies); urticaria (which is often characterized,for example, by IgG and IgM antibodies to IgE); atopic dermatitis (whichis often characterized, for example, by IgG and IgM antibodies to IgE);asthma (which is often characterized, for example, by IgG and IgMantibodies to IgE); inflammatory myopathies; and other inflammatory,granulomatous, degenerative, and atrophic disorders.

Also included are methods of modulating hematopoiesis and relatedconditions. Examples of hematopoietic processes that may be modulated bythe AspRS polypeptides of the invention include, without limitation, theformation of myeloid cells (e.g., erythroid cells, mast cellsmonocytes/macrophages, myeloid dendritic cells, granulocytes such asbasophils, neutrophils, and eosinophils, megakaryocytes, platelets) andlymphoid cells (e.g., natural killer cells, lymphoid dendritic cells,B-cells, and T-cells). Certain specific hematopoietic processes includeerythropoiesis, granulopoiesis, lymphopoiesis, megakaryopoiesis,thrombopoiesis, and others. Also included are methods of modulating thetrafficking or mobilization of hematopoietic cells, includinghematopoietic stem cells, progenitor cells, erythrocytes, granulocytes,lymphocytes, megakaryocytes, and thrombocytes.

The methods of modulating hematopoiesis may be practiced in vivo, invitro, ex vivo, or in any combination thereof. These methods can bepracticed on any biological sample, cell culture, or tissue thatcontains hematopoietic stem cells, hematopoietic progenitor cells, orother stem or progenitor cells that are capable of differentiating alongthe hematopoietic lineage (e.g., adipose tissue derived stem cells). Forin vitro and ex vivo methods, stem cells and progenitor cells, whetherof hematopoietic origin or otherwise, can be isolated and/or identifiedaccording to the techniques and characteristics described herein andknown in the art.

In other embodiments, the AspRS compositions of the invention may beused to modulate cellular proliferation and/or survival and,accordingly, for treating or preventing diseases, disorders orconditions characterized by abnormalities in cellular proliferationand/or survival. For example, in certain embodiments, the AspRScompositions may be used to modulate apoptosis and/or to treat diseasesor conditions associated with abnormal apoptosis. Apoptosis is the termused to describe the cell signaling cascade known as programmed celldeath. Various therapeutic indications exist for molecules that induceapoptosis (e.g. cancer), as well as those that inhibit apoptosis (i.e.stroke, myocardial infarction, sepsis, etc.). Apoptosis can be monitoredby any of a number of available techniques known and available in theart including, for example, assays that measure fragmentation of DNA,alterations in membrane asymmetry, activation of apoptotic caspasesand/or release of cytochrome C and AIF.

Illustrative diseases associated with increased cell survival, or theinhibition of apoptosis include, but are not limited to, cancers (suchas follicular lymphomas, carcinomas, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis,autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenicpurpura, and rheumatoid arthritis) and viral infections (such as herpesviruses, pox viruses and adenoviruses), inflammation, graft vs. hostdisease (acute and/or chronic), acute graft rejection, and chronic graftrejection.

Further illustrative diseases or conditions associated with increasedcell survival include, but are not limited to, progression and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (for example, acute lymphocytic leukemia,acute myelocytic leukemia, including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(for example, chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia), myelodysplastic syndrome polycythemia vera,lymphomas (for example, Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Illustrative diseases associated with increased apoptosis include, butare not limited to, AIDS (such as HIV-induced nephropathy and HIVencephalitis), neurodegenerative disorders (such as Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, cerebellar degeneration and brain tumor or prior associateddisease), autoimmune disorders such as multiple sclerosis, Sjogren'ssyndrome, Graves' disease, Hashimoto's thyroiditis, autoimmune diabetes,biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis,systemic lupus erythematosus, immune-related glomerulonephritis,autoimmune gastritis, thrombocytopenic purpura, and rheumatoidarthritis, myelodysplastic syndromes (such as aplastic anemia), graftvs. host disease (acute and/or chronic), ischemic injury (such as thatcaused by myocardial infarction, stroke and reperfusion injury), liverinjury or disease (for example, hepatitis related liver injury,cirrhosis, ischemia/reperfusion injury, cholestosis (bile duct injury)and liver cancer), toxin-induced liver disease (such as that caused byalcohol), septic shock, ulcerative colitis, cachexia, and anorexia.

In still further embodiments, the compositions of the invention may beused in the treatment of neuronal/neurological diseases or disorders,illustrative examples of which include Parkinson's disease, Alzheimer'sdisease, Pick's disease, Creutzfeldt-Jacob disease, Huntington's chorea,alternating hemiplegia, amyotrophic lateral sclerosis, ataxia, cerebralpalsy, chronic fatigue syndrome, chronic pain syndromes, congenitalneurological anomalies, cranial nerve diseases, delirium, dementia,demyelinating diseases, dysautonomia, epilepsy, headaches, Huntington'sdisease, hydrocephalus, meningitis, movement disorders, muscle diseases,nervous system neoplasms, neurocutaneous syndromes, neurodegenerativediseases, neurotoxicity syndromes, ocular motility disorders, peripheralnervous system disorders, pituitary disorders, porencephaly, Rettsyndrome, sleep disorders, spinal cord disorders, stroke, sydenham'schorea, tourette syndrome, nervous system trauma and injuries, etc.

Furthermore, additional embodiments relate to the use of thecompositions of the invention in the treatment of metabolic disorderssuch as adrenoleukodystrophy, Krabbe's disease (globoid cellleukodystrophy), metachromatic leukodystrophy, Alexander's disease,Canavan's disease (spongiform leukodystrophy), Pelizaeus-Merzbacherdisease, Cockayne's syndrome, Hurler's disease, Lowe's syndrome, Leigh'sdisease, Wilson's disease, Hallervorden-Spatz disease, Tay-Sachsdisease, etc. The utility of the compositions of the invention inmodulating metabolic processes may be monitored using any of a varietyof techniques known and available in the art including, for example,assays which measure adipocyte lipogenesis or adipocyte lipolysis.

In more specific embodiments of the invention, the AspRS compositions ofthe invention may be used to modulate cellular signaling, for example,via cell signaling proteins (e.g., Akt). Cell signaling may be monitoredusing any of a number of well known assays. For example, the inductionof general cell signaling events can be monitored through alteredphosphorylation patterns of a variety of target proteins. Detection ofcell signaling activities in response to treatment of cells with AspRSpolypeptides therefore serves as an indicator of distinct biologicaleffects. Target proteins used for this assay may be selected so as toencompass key components of major cellular signaling cascades, therebyproviding a broad picture of the cell signaling landscape and itstherapeutic relevance. Generally, such assays involve cell treatmentwith AspRS polypeptides followed by immunodetection with antibodies thatspecifically detect the phosphorylated (activated) forms of the targetproteins.

Illustrative target proteins used for monitoring therapeuticallyrelevant cell signaling events may include, but are not limited to: p38MAPK (mitogen-activated protein kinase; activated by cellular stress andinflammatory cytokines; involved in cell differentiation and apoptosis);SAPK/JNK (stress-activated protein kinase/Jun-amino-terminal kinase;activated by cellular stresses and inflammatory cytokines); Erk1/2,p44/42 MAPK (mitogen-activated protein kinase Erk1 and Erk2; activatedby wide variety of extracellular signals; involved in regulation of cellgrowth and differentiation); and Akt (activated by insulin and variousgrowth or survival factors; involved in inhibition of apoptosis,regulation of glycogen synthesis, cell cycle regulation and cellgrowth). General phosphorylation of tyrosine residues may also bemonitored as a general indicator of changes in cell signaling mediatedby phosphorylation.

Of course, it will be recognized that other classes of proteins, such ascell adhesion molecules (e.g., cadherins, integrins, claudins, catenins,selectins, etc.) and/or ion channel proteins may also be assayed formonitoring cellular events or activities modulated by the compositionsof the invention.

In other specific embodiments of the invention, the AspRS compositionsof the invention may be used to modulate cytokine production by cells,for example, by immune cells such as monocytes and/or leukocytes.Cytokine production may be monitored using any of a number of assaysknown in the art (i.e., RT-PCR, ELISA, ELISpot, flow cytometry, etc.).Generally, such assays involve cell treatment with AspRS polypeptidesfollowed by detection of cytokine mRNA or polypeptides to measurechanges in cytokine production. Detection of increases and/or decreasesin cytokine production in response to treatment of cells with AspRSpolypeptides therefore serves as an indicator of distinct biologicaleffects. AspRS polypeptides of the invention may induce, enhance, and/orinhibit an immune or inflammatory response by modulating cytokineproduction. For example, AspRS polypeptides and compositions of theinvention may be used to alter a cytokine profile (i.e., type 1 vs. type2) in a subject. Illustrative cytokines that may measured for monitoringbiological effects of the AspRS compositions include, but are notlimited to IL-1ra, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23 TGF-β, TNF-α, IFN-α,IFN-β, IFN-γ, RANTES, MIP-1α, MIP-1β, MCP-1, GRO-α, GM-CSF, G-CSF, etc.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of noncritical parameters thatcould be changed or modified to yield essentially similar results.

EXAMPLES Example 1 Generation of Human Aspartyl-tRNA Synthetase (AspRS)Fragments

Full-length recombinant human AspRS having an amino acid sequence as setforth in SEQ ID NO: 1 was expressed and purified from E. coli usingnickel IMAC chromatography. To generate fragments of AspRS by controlledproteolysis, the full-length protein was treated with 42 nM humanneutrophil elastase for 30 minutes before separation of the fragments bySDS-PAGE run in 4-12% MOPS or 12% MES buffer (FIGS. 1C and D).Digestions run on SDS-PAGE gels in 4-12% MOPS revealed only a singleprotein fragment at approximately 19 kDa (FIG. 1C), while digestions runon SDS-PAGE gels in 12% MES buffer revealed at least three additionalsmaller peptide fragments between 3 and 6 kDa (FIG. 1D).

Example 2 AspRS Fragments Activate Akt in Endothelial Cells

Pools of AspRS fragments were generated by adding 42 nM neutrophilelastase to 2 ug full-length recombinant AspRS for 30 minutes at 37° C.Reactions were stopped by the addition of alpha 1-antitrypsin (SerpinA1) in 10-fold excess of the protease. Bovine aortic endothelial cells(bAEC) were treated with pools of 50 nM full-length AspRS proteinuncleaved or cleaved with neutrophil elastase. Cells were incubated withAspRS fragments for 10 and 15 minutes, harvested and subjected toWestern blotting with an antibody that specifically recognizes only thephosphorylated (activated) form of the signaling molecule Akt. Thistreatment resulted in strong, reproducible activation of Akt viaphosphorylation (FIGS. 2A and 2B). This effect is significant due to therole of Akt in the regulation of apoptosis, glycogen synthesis, cellcycle regulation, and cell growth.

Example 3 Identification of Neutrophil Elastase Cleavage Sites on AspRS

Fragments generated by cleavage with neutrophil elastase (FIG. 1D) wereanalyzed using LC/MS/MS to determine accurate masses for each fragment.In addition, individual fragments were excised from an SDS-PAGE gel runin 4-12% MOPS or 12% MES buffer and subjected to in-gel trypsindigestion followed by LC/MS/MS analysis to identify the portion of thefull-length protein from which the fragment was generated and toidentify non-trypsin cleavage sites that could be attributed toneutrophil elastase. The identity of these peptide boundaries issummarized in Table 2.

TABLE 2 AspRS peptide boundaries Whole mass Protease N-term. C-termFragment (Da) used boundary boundary D1 19437 elastase 1 154 18370 D221590 elastase 1 174 D3 4367 elastase 1 31 4468 D4 3309 elastase 399 425D5 2517 elastase 413 476 D6 3479 elastase 397 425

Example 4 AspRS Fragment Increases TNF-α Secretion from PBMCs

Peripheral blood mononuclear cells (PBMCs) from healthy donors weretreated with 100 nM doses of full-length AspRS protein and a fragment ofAspRS, D1 (Table 2), for 24 hours. EMAPII(Endothelial-monocyte-activating polypeptide II), which is known toincrease TNF-α secretion from PBMCs, was used as a positive control. Anincrease in TNF-α secretion was observed in response to the full-lengthAspRS and this increase was similar in magnitude to that observed forthe EMAPII positive control. Unexpectedly, however, the D1 fragment ofAspRS induced TNF-α secretion at a level nearly 6-fold higher than thatobserved for either full length AspRS or the EMAPII positive control(FIG. 3). Thus, the D1 fragment of AspRS has a novel function that islargely masked within the full length protein.

Example 5 AspRS Fragment D1 Induces In Vitro Secretion of CytokinesDistinct from Full Length AspRS

Full length AspRS (100 nM) or a fragment of AspRS, D1 (100 nM), wereincubated with 1×10⁶ Peripheral Blood Mononuclear Cells (PBMC) for 24hours. After 24 hours of incubation, supernatants were harvested, snapfrozen in liquid nitrogen and then analyzed for multiple cytokines.Supernatants were measured for 27 distinct cytokines and compared tobuffer-treated PBMC supernatants. Error bars are representative of 2biological replicates. As shown in FIG. 4, AspRS fragment D1 showed alarge stimulation of numerous cytokines above and beyond stimulationsobserved with full length AspRS (e.g., IL1-13, IL-6, IL-8, IL-10,IL-12p40, MIP1-α, MIP-1β, GRO-α, MCP-1, and IL-1ra).

Example 6 AspRS Fragment Induces CD71 Marker Upregulation in Monocytes

Peripheral blood mononuclear cells (PBMC's) were isolated from a normalblood donor. 1.5×10⁶ PBMC's were treated with a 200 nM dose of the AspRSfragment D1 (consisting of the first 154 amino acids of the full lengthprotein) for 24 hours. PBMC's treated with 10 μg/mL of the plant lectinphytohemagglutinin (PHA) served as a positive control. As shown in FIG.5, upregulation of the CD71 proliferation marker was seen in theD1-treated gated monocytes after staining with an anti-CD71 antibody(Beckton-Dickinson) and analyzing the samples by flow cytometry. Therewas no significant increase in CD71 upregulation in the gated lymphocytepopulation of the same samples. Thus, D1 has a cell type specificability to activate monocytes in a PBMC mixture.

Example 7 AspRS Fragment Increases TNF-α Secretion from Monocytes andMacrophages

Both monocyte (THP-1) and macrophage (RAW 264.7) cell lines were treatedwith C-terminally tagged D1 (C-D1) or full length AspRS (C-DRS) at 100nM. Supernatant was collected at 2, 4, 8 and 24, hours and then analyzedfor TNF-α secretion. As shown in FIG. 6, the maximal amount of TNF-αsecretion after treatment with C-D1 was seen between 2 and 4 hours, butthen decreased at 8 and 24 hours. TNF-α secretion following treatmentwith C-DRS was negligible at all time points examined. The increase inTNF-α secretion following treatment with C-D1 was dose-dependent. Inaddition, treatment of cells with 100 nM, 50 nM, 25 nM, 12.5 nM, and 6nM C-D1, N-D1, and C-DRS for 4 hours demonstrated that only C-D1treatment increased TNF-α secretion.

Example 8 DRS Fragment D1 Induces Chemotaxis of a Macrophage Cell Line

To assess cell migration in vitro, 24-well Transwell chambers withpolycarbonate membranes (5 μm pore size, Costar) were coated with 0.5mg/ml gelatin in PBS and allowed to air dry. Detached RAW 264.7 cells(mouse monocyte/macrophage cell line) were washed once with fresh DMEMand suspended into 2×10⁷ cells/ml with 0.1% BSA/DMEM. Full-length AspRS(DRS) or D1 was diluted with 0.1% BSA/DMEM into differentconcentrations. RAW 264.7 cells were added to the upper chamber at 2×10⁶cells in 100 μl per well. The lower chambers were filled with 500 μl perwell of media containing DRS or D1. After 24 hours at 37° C., calcein AM(Invitrogen) was added to lower chambers at a final concentration of 8μM to stain migrated cells. Following a 30 minute incubation, cells thathad not migrated were removed from the upper surface of the Transwellmembrane with a cotton swab. Migrating cells on the lower membranesurface were counted under fluorescence microscope in high power fields.As shown in FIG. 7, D1 induced migration in a dose dependent manner,whereas little to no migration was stimulated by full length AspRS atthe same concentrations.

Example 9 AspRS Fragment Induced TNF-α Secretion Macrophages can beInhibited by U0126

Macrophage (RAW 264.7) cells were pre-treated with the small-moleculeinhibitors U0126 or LY294022 at 100 nM for one hour followed bytreatment with D1 at 50 nM or LPS at 1 ng/ml for an additional 4 hours.Supernatant was collected and analyzed for TNF-α secretion. As shown inFIG. 8, secretion of TNF-α was inhibited by U0126 in D1 and LPS treatedcells. However, LY294022 only inhibited TNF-α secretion in LPS treatedcells.

Example 10 AspRS Fragment D1 Inhibits VEGF-Induced Angiogenesis

The purpose of this experiment was to evaluate the anti-angiogenicactivity of the D1 fragment of AspRS. D1 protein was directlyincorporated into Matrigel® plugs to determine its' ability to inhibitVEGF-induced angiogenesis in a Modified Matrigel® Plug Assay. Briefly,female NCR Nude mice (8 mice/group) were obtained that weighed 21-25 gon Day 1 of the experiment. Air pouches were generated in test animalsby injecting 1 ml air into the subcutaneous space between the scapulaeon Days 1, 4, and 6 using a 27-gauge needle. On Day 7, 0.5 ml Matrigel®(VWR) containing VEGF (Cell Sciences)+Saline, VEGF+Sutent (PfizerPharmaceuticals), or VEGF+D1 protein was injected into the previouslycreated air pouches. On Day 13 (6 days after implant) animals wereeuthanized and the Matrigel® plugs were excised, photographed, andweighed. The primary endpoint used to evaluate activity was thehemoglobin content per mg of wet Matrigel® plug weight. As Shown in FIG.9, D1 caused an inhibition of VEGF-induced angiogenesis.

Example 11 C-Terminally Tagged AspRS Fragment Induced TNF-α Secretion inMonocytes

Monocyte (THP-1) cells were treated with C- or N-terminally tagged D1 orfull length AspRS at 100 nM for four hours. After which, supernatant wascollected and analyzed for TNF-α secretion. As shown in FIG. 10,induction of TNF-α secretion was the greatest in cells treated withC-terminally tagged D1. N-terminally tagged D1 induced a much smallerresponse, indicating the N-terminus region of the D1 fragment likelyplays an important role in its cytokine activity. All other treatmentgroups had significantly lower induction of TNF-α secretion.

Example 12 AspRS Fragment D1 Contains a Mammalian-Specific Domain ofHuman Drs

As shown in FIG. 11, a 32 amino acid peptide is found only at theN-terminus of mammalian DRS and is not found in yeast DRS. This regionof the protein is dispensable for canonical tRNA synthetaseaminoacylation activity and is predicted to contain a putativeamphiphilic helix (reported in Jacobo-Molina and Yang (1989), Escalanteand Yang, JBC (1992)). Based on the observed importance of theN-terminus of D1 in relation to its cytokine activity, this uniqueregion may be an important mediator of the cytokine activity reportedhere for D1.

Example 13 Identification of Endogenous D1 Fragment from Macrophages

As illustrated in FIG. 12A, a fragment of AspRS was detected in a mousemacrophage cell line (RAW264.7) using LC/MS/MS proteomics analysis. FIG.12A shows the steps by which RAW264.7 mouse macrophages were subjectedto SDS-PAGE analysis; protein bands were cut out and analyzed by LCMS/MS, and an N-terminal fragment of AspRS was identified as D1. Thismass spectral analysis revealed that the D1 fragment comprises theN-terminal portion of the 501 residue monomer unit of the AspRShomodimer (consisting approximately of residues 1-171 of full-lengthAspRS). The D1 fragment includes the anticodon-binding domain of humanAspRS (see FIG. 12B), and has structural similarity to theEMAPII-cytokine that contains a highly similar OB-fold domain. TheEMAPII cytokine is found as a distinct domain in p43 (a protein that isbound in the multi-tRNA synthetases complex of mammalian cells) where,under apoptotic conditions, it is resected and secreted to serve as animmunomodulatory cytokine. A similar EMAPII-like domain exists in theC-terminal region of human TyrRS. However, in contrast to EMAP-II andthe homologous domains found in TyrRS and p43, D1 has a unique 22 aminoacid extension at the N-terminus that is found only in higher eukaryotesand forms an amphiphilic helix.

Example 14 Structural Analysis of AspRS

To better understand the structure and physiological origin of D1,native human AspRS was crystallized and its 3-dimensional structure wasdetermined to a resolution of 1.9 Å (see FIG. 12C). The part of thestructure corresponding to D1 forms a separate OB-fold-containingdomain, while the C-terminal catalytic domain quite resembles that ofyeast and bacterial AspRS. The linker encompassing residues 154 and 182that connect the D1 fragment and the catalytic domain was structurallydisordered, suggesting its high flexibility. The flexibility of thislinker region and its apparent accessibility to proteases, suggestedthat its cleavage by endogenous proteases should liberate D1 from nativeAspRS. Treatment of recombinant native human AspRS with PMN elastaseconfirmed this expectation by cleavage and clean release of D1 atresidue 154 (see Example 3).

Example 15 AspRS Fragment D1 Induces In Vivo and In Vitro Secretion ofCytokines and Binds to Immune Cells

Macrophages are key players in innate immunity, and produce and secretea large number of protein cytokines including those involved in cellularmetabolism and inflammation. To probe the possible connection betweenthe D1 fragment of AspRS and inflammation, D1 protein (10 mg/kg) wasinjected intravenously into healthy mice, and changes were measured ininflammatory cytokines (both pro- and anti-inflammatory) secreted intothe bloodstream relative to vehicle controls. Because human and mouseAspRS and D1 have 96.8% sequence identity, recombinant human AspRS andD1 was used for all studies.

FIG. 13A shows in vivo TNF-α and IL-10 serum levels from mice injectedintravenously with 10 mg/kg D1. Mice show an increase in TNF-α after 2hours that is quickly cleared by 6 hours while IL-10 levels continue toincrease.

To confirm these in vivo results, PBMCs representing a mixture of bothmonocytes and lymphocytes and isolated from human donors were alsoexposed to the D1 protein in vitro (as well as the full-length AspRSprotein) and tested the media for the secretion of either TNF-α or IL-10in response to treatment. Similar to the effects observed in vivo,treatment with D1 resulted in secretion of both TNF-α and IL-10 fromthis mixed cell population (FIG. 13B). The effects were specific to D1and were not seen with native AspRS, illustrating the effects ofisolating this N-terminal domain from the parent tRNA synthetase by theprocess of resection.

To investigate which cells within the PBMC mixture were targeted by D1,its binding to the different subpopulations of cells within the mixturewas analyzed using flow cytometry. As shown in FIG. 13C, robust bindingof D1 to monocytes was observed, with almost 100% of monocytes in themixture bound by a D1 molecule. In contrast, no binding of native AspRSto monocytes was detected (data not shown). Binding of the D1 proteinwas also observed for a subset of lymphocytes (˜14%). Further analysisof the bound lymphocyte population revealed that D1 binding occurs onboth B cells (˜80% of total B cells were bound) and T cells (˜20% oftotal T cells were bound) (see FIG. 13C, inset). For both monocytes andlymphocytes, the effects were specific to D1 and were not seen withnative AspRS (data not shown). These data support a role for D1 indirectly binding and perhaps modulating cells involved in the immuneresponse.

Example 16 D1 Signals Through Nuclear Factor-kB (NF-κB)

Nuclear factor-kB (NF-kB) is a transcription factor thought to play animportant role in onset of inflammation through stimulatingtranscription of pro-inflammatory cytokines (like TNF-α) and, during theresolution of inflammation, to then stimulate expression ofanti-inflammatory cytokines like IL-10. NF-κB also plays a central rolein directing cellular responses to many stimuli, including oxidativestress, viral and bacterial pathogens, and inflammatory cytokines. Theeffects of D1 on the activation of NF-κB in macrophages were thereforeinvestigated.

For this experiment, RAW-Blue cells encoding an NF-κB-inducible secretedembryonic alkaline phosphatase reporter gene were incubated with PBS,D1, or full-length AspRS. As shown in FIG. 14A, the treated cells showeda strong dose-dependent activation of NF-κB with D1, as compared to thelack of activation by full-length AspRS or PBS.

Example 17 D1 Binds to and Signals Through TLR2 and TLR4

NF-κB can be triggered through a number of macrophage cell surfacereceptors including the pattern-recognizing toll-like receptors (TLRs).To investigate the potential link to the TLR receptor family, 7different HEK293 cell lines were stably co-transfected with NF-κBinducible reporter genes (encoding secreted embryonic alkalinephosphatase) and genes encoding a panel of separate toll-like receptors(TLR2, TLR3, TLR4, TLR 5, TLR 7, TLR 8, and TLR9). As shown in FIG. 14B,D1-induced activation of NF-κB was observed only through TLR2 or TLR4and not TLRs 3, 5, 7, 8, or 9.

Flow cytometry experiments were utilized to establish whether D2 bindsto TLR2 and/or TLR4. In these experiments, V5-tagged D1 (100 nM) orAspRS (100 nM) was incubated with HEK293 cells stably expressing TLR2 orTLR4. Empty vector transfected HEK293 cells served as the null bindingcontrol. Binding was assessed by FITC-V5 detection using flow cytometry.FIG. 14C shows that D1 bound strongly to stably transfected HEK cellsover-expressing TLR2 or TLR4, but bound much less so to HEK cells thatwere transfected with vector alone, and which did not express TLR2 orTLR4.

Example 18 The Amphiphilic Helix in D1 Activity

Prior work established LPSs as ligands for TLR2 and 4. Indeed, a lipid Aagonist (OM174) ligand for TLR2 and 4 demonstrated similar effects invivo to what has been observed for D1, namely, transient release ofTNF-α (1-2 hours) and subsequent increases in IL-10 secretion. D1encodes an EMAP-II-like OB-fold. Like D1, the EMAP-II domain, whenreleased by proteolytic cleavage from p43, stimulates secretion of TNF-αfrom monocytes. The EMAP-II domain also shows additional activity onneutrophils (stimulating migration and secretion of myeloperoxidase).D1, however, did not act on neutrophils (data not shown). Onedistinction between EMAP-II and D1 is the unique amphiphilic helixcontained within the first 22 amino acids of D1. The role of theamphiphilic helix in D1 activity was therefore investigated.

Initially, the entire amphiphilic helix region of 22 amino acids wasdeleted from D1 to give Δ22 D1. Certain point mutations were alsogenerated. For instance, as an amphiphilic helix, the human D1N-terminal helix contains positively charged residues on one side of thehelix and negatively charged residues on the other. D1 of lowereukaryotes has a slightly longer helix that is positively charged onboth sides. The positively charged residues of this helix have beendemonstrated to strengthen tRNA binding, with the consensus sequenceLSKKXLKKXXK (SEQ ID NO:6) being particularly important. The evolution ofthis helix from a positively charged to an amphiphilic helix (throughthe progression from lower to higher eukaryotic AspRSs) occurs via aconcerted switch of 3 highly conserved residues to create a cluster ofnegative charges on one side of the helix that is strictly conserved inhigher eukaryotes (FIGS. 4A and B). It was hypothesized that thesenegatively charged residues in particular, when in the context of theEMAP-II-like OB-fold, may be contribute to the activity of the novel22-amino acid helix that is appended to D1. To explore this possibility,substitutions were made at the 3 conserved residues of the negativelycharged higher eukaryotic cluster (E12, E16, D19) (see FIG. 15B) toswitch them back to the lower eukaryotic form (E12S, E16K, D19K) withits positively charged cluster (SKK D1).

PBMCs were then treated with 50 nm D1, full-length AspRS, the Δ22mutant, and charge mutants AAA and SKK. Compared to intact D1, Δ22 D1induced very little TNF-α or IL-10 release from PBMCs (see FIG. 15C).The AAK and SKK D1 mutants also had reduced (˜4-fold for SKK) activityfor both TNF-α and IL-10 secretion, as compared to D1. Theseobservations suggest a role for the N-terminal amphiphilic helix inreceptor binding.

In further support of this conclusion, an N-V5-D1, with the V5-tag atthe N-rather than the C-terminus, was also constructed and tested inboth TNF-α release and binding assays. The N-V5-D1 was unable to bind orinduce TNF-α secretion from PBMCs (data not shown). Thus, the activityof D1 can be reduced with a peptide fusion at the N-terminus, furthersupporting the role of the N-terminal amphiphilic region in D1 activity.

Example 19 D1 Activity is not Due to Endotoxin Contamination

The recombinant D1 used in these studies was purified from E. coli andshown to have an LPS-containing bacterial endotoxin level of less than12 EU/mg. Nonetheless, LPS is a strong stimulant of TLR2 and TLR4signaling, and experiments were performed to remove any possibility oftrace endotoxin being responsible for the results seen with D1. For thispurpose, a gene encoding D1 with a secretion sequence was expressed intransfected HEK293 cells. Conditioned media containing secreted D1 wascollected, concentrated, and incubated with PBMCs.

As shown in FIG. 16A, these media stimulated secretion of TNF-α, whilemedia from cells transfected with vector alone did not stimulatesecretion. Further, D1-stimulated TNF-α release was unaffected bypolymyxin B, a known inactivator of endotoxin (see FIG. 16B). Incontrast, as also shown in FIG. 16B, LPS-stimulated activation ofsecretion of TNF-α was completely blocked by polymyxin B. As shown inFIG. 16C, D1 treated with proteinase K, which completely digestsproteins but not endotoxin, resulted in complete abrogation of TNF-αactivity in PBMCs. D1 activity is therefore not due to endotoxincontamination.

Example 20 Δ22 AspRS In Vivo Mouse Knock-in Experiments

Because the Δ22 variant of AspRS does not stimulate TNF-α secretionthrough TLR2 and TLR4, as shown above, the generation of a Δ22 AspRSknock-in mouse allows examination of the physiological effects of theN-terminus of AspRS without compromising the canonical and essentialaminoacylation activity of AspRS. Initial experiments focus oneexamining the potential protective effects of removal of the Δ22 regionof AspRS, which has been shown to contribute to AspRS activity as a TLR2and TLR4 endogenous ligand.

An endotoxic shock experiment is performed to test whether mice with aΔ22 AspRS knock-in are more resistant to endotoxic shock because oftheir lack of an endogenous ligand for TLR2 and TLR4. In thisexperiment, wild-type and Δ22 AspRS knock-in mice are injectedintraperitoneally with LPS (1 μg/mouse) in combination with D-GalN (20mg) in a saline solution of 200 μL per dose. This is a commonly usedmodel for endotoxic shock or sepsis that results in near completelethality in wild-type mice (see Car et al., J Exp Med, 179:1437-44,1994). It is believed that by removing an endogenous pro-inflammatorytoll-like receptor ligand (i.e., the AspRS region represented by Δ22)the mice should be resistant to endotoxic shock induced lethality.

An LPS tolerance experiment is performed to test whether macrophagesfrom Δ22 AspRS mice are less tolerant to LPS stimulation due to the lackof a desensitization of toll-like receptor signaling. Macrophages fromthese knock-in mice should not have been exposed to the pro-inflammatoryeffects of an endogenous TLR2 & TLR4 ligand (i.e., the AspRS regionrepresented by Δ22). To examine this possibility, wild-type and Δ22AspRS knock-in mouse peritoneal macrophages are stimulated ex vivo withLPS (100 ng/mL) for 24 hrs, which results in an activation andproduction of cytokines that can be analyzed by ELISA (see Sato et al.,J Immunol. 165:7096-101, 2000). Macrophages from the Δ22 AspRS miceshould demonstrate a stronger response to LPS due to the lack of apro-inflammatory signal that otherwise contributes to the induction oftolerance.

As noted, the disclosure above is descriptive, illustrative andexemplary and is not to be taken as limiting the scope defined by theappended claims which follow.

1-37. (canceled)
 38. A pharmaceutical composition, comprising aphysiologically-acceptable carrier and an aspartyl-tRNA synthetase(AspRS) polypeptide consisting of 50-200 amino acids in length thatcomprises (a) amino acid residues 1-154, 1-171, or 1-174 of SEQ ID NO:1, or (b) an active variant having at least 95% sequence identity to thepolypeptide of (a), wherein the AspRS polypeptide comprises anamphiphilic helix, and wherein the composition has an LPS-containingbacterial endotoxin level of less than about 12 EU/mg.
 39. Thepharmaceutical composition of claim 38, wherein the active variant hasat least 98% identity to the polypeptide of (a).
 40. The pharmaceuticalcomposition of claim 38, wherein the AspRS polypeptide consistsessentially of amino acid residues 1-154, 1-171, or 1-174 of SEQ IDNO:1.
 41. The pharmaceutical composition of claim 40, wherein the AspRSpolypeptide consists essentially of amino acid residues 1-154 of SEQ IDNO:1.
 42. The pharmaceutical composition of claim 38, wherein the activevariant comprises at least one substitution in the amphiphilic helixwhich modulates the ability of the variant to induce IL-10 cytokinerelease from PBMCs relative to an AspRS polypeptide of claim 1(a). 43.The pharmaceutical composition of claim 42, wherein the at least onepoint mutation is at position E12, E16, or D19.
 44. The pharmaceuticalcomposition of claim 38, wherein the AspRS polypeptide is modified bypegylation.
 45. The pharmaceutical composition of claim 38, wherein theAspRS polypeptide is fused to a heterologous fusion partner.
 46. Thepharmaceutical composition of claim 45, wherein the heterologous fusionpartner comprises an Fc fragment.
 47. The pharmaceutical composition ofclaim 38, wherein the composition is formulated for oral, parenteral,intravenous, intranasal, inhalation, aerosol, intracranial, orintramuscular administration.
 48. The pharmaceutical composition ofclaim 38, wherein the anti-inflammatory activity is induction ofanti-inflammatory cytokine production or activity, binding to toll-likereceptor(s), or both.
 49. A method of treating an inflammatory orautoimmune disease in a subject in need thereof, comprisingadministering to the subject a pharmaceutical composition of claim 38.